Substituted 5-(pyrazin-2-yl)-1H-pyrazolo [3, 4-b] pyridine and pyrazolo [3, 4-b] pyridine derivatives as protein kinase inhibitors

ABSTRACT

Substituted 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine and pyrazolo[3,4-b]pyridine derivatives according to formula I, II and VII, and methods for making same, which are inhibitors of constitutively activated Tyrosine Kinase-Like (TKL), CMGC protein kinases family members and can be useful in the treatment of Parkinson&#39;s disease, Alzheimer&#39;s disease, Down&#39;s Syndrome, Huntington&#39;s disease, other neurodegenerative and central nervous system disorders, cancer, metabolic disorders and inflammatory diseases. Also disclosed are pharmaceutical compositions including the compounds and methods of inhibiting wild type and/or mutated protein kinase activities of these families and the treatment of disorders associated therewith using compounds and pharmaceutical compositions including the compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 13/435,327, filed Mar. 30, 2012 and claims the benefit of U.S. Provisional Patent Application No. 61/469,274, filed Mar. 30, 2011, the entire disclosures of which are incorporated herein by reference.

INCORPORATION BY REFERENCE

The sequence listing submitted in the copies of the compact discs COPY 1 and COPY 2 filed in connection with this application and the files contained on each disc, each entitled “Sequence Listing”, each 372 KB, each created Jun. 8, 2012, are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds useful as protein kinase inhibitors, specifically LRRK2 (Leucine-Rich Repeat Kinase-2) inhibitors, pharmaceutically acceptable compositions including such compounds and methods of using said compounds in the treatment of various diseases and or disorders.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) and Alzheimer's disease (AD) are the most common neurodegenerative disorders in the adult population. Parkinson's disease is characterized by slow movement, resting tremor, rigidity, bradykinesia and postural instability. The disease is characterized pathologically by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of intracytoplasmic aggregates called Lewy bodies in surviving neurons of the brainstem. The incidence of PD increases with age; it has been estimated 0.3% of those age 50 are afflicted, the number increasing to 4.3% by age of 85. No significant treatment is available for PD.

Alzheimer's disease is a fatal neurodegenerative disorder which results in progressive memory loss and behavioral abnormalities for which there is currently no cure. AD is characterized by the progressive formation of insoluble amyloid plaques and fibrillary tangles. These plaques primarily of consists of an aggregated αβ (Amyloid-β) peptide that is formed by the proteolytic processing of amyloid precursor protein (APP) by β-site cleaving enzyme (BACE1). Current approaches targeting these proteins to treat AD do not arrest the underlying disease process and largely treat only the symptoms. Therapeutics to either slow or reverse the neuronal loss and the underlying cognitive decline are urgently needed.

The human gene LRRK2 (Leucine-Rich Repeat Kinase-2)(SEQ ID NO 62) encodes an enzyme also known as LRRK2 or dardarin. LRRK2 is a 144 Kb with 51 exons encoding 2527 amino acids. The LRRK2 protein domain consists of Ras belonging to the Ras/GTPase family, COR, LRR, a Leucine-Rich Repeat, consisting of MAPKKK kinase catalytic domain a (22-28 amino acid motif) directly involved in Ser/Thr phosphorylation, a WD40 domain and ankyrin repeats. LRRK2 is expressed in all tissues at low levels and in neurons of the brain regions including the cortex, striatum, hippocampus, and cerebellum and in dopaminergic neurons of the substantia nigra. Although the expression levels of LRRK2 in the dopaminergic neurons of the SNpc are very low, its expression level has been detected in Lewy neurites and in Lewy bodies of sporadic PD.

LRRK2 (SEQ ID NO 62) is reported to be the most prevalent cause for autosomal dominant PD. Overlapping neurodegenerative pathologies have been described in PD patients with Alzheimer's disease with LRRK2 mutations. A common G2019S mutation (SEQ ID NO 28) accounts for a significant proportion of sporadic PD in some populations and the location of the LRRK2 gene on chromosome 12q12 that causes late onset AD. More than 20 mutations have been reported in the gene encoding LRRK2 (SEQ ID NO 62) which have been linked recently with autosomal-dominant Parkinsonism that is clinically indistinguishable from typical, idiopathic, late-onset PD. Some of these mutations, R1441C, R1441G, Y1699C and G2019S, are amino acids conserved. Recent study demonstrates that LRRK2 R1628P (SEQ ID NO 29) variant is associated with a two-fold risk of AD.

SUMMARY OF THE INVENTION

Recent studies showed pathogenic mutations of LRRK2 (SEQ ID NO 62) increased LRRK2 kinase activity with autophosphorylation, suggesting a gain-of-function mechanism. Recent findings suggest that S2031 and T2032 are critical residues required for LRRK2 autophosphorylation, and T2035 is important for its catalytic activity. Dimeric LRRK2 undergoes intramolecular autophosphorylation for its kinase activity. PD patients having LRRK2 mutations are subject to neuronal degeneration in the brain, due to induced cell toxicity in multiple cell lines and rodent primary neurons with reduction of cell viability ranging from 10-40%. Expression of G2019S decreases neuronal cell viability 2 to 5 fold. Mice hybridization studies reveal that expression of LRRK2 (SEQ ID NO 62) overexpressed in the brain is higher within regions of the basal ganglia and directly associated with motor dysfunction in PD. It has been postulated that LRRK2 may be involved in some overlapping neurodegenerative pathways in AD.

The protein LRRK2 (SEQ ID NO 62) is a therapeutic target for the treatment of PD pathogenesis and the compounds of the invention of Formula I, II and VII associated matter are capable of inhibiting one or more protein kinases, more particularly LRRK2 (SEQ ID NO 62) and more particularly mutated LRRK2. Targeting of LRRK2 may be employed to treat PD and AD pathologies. The disclosed compounds are potent and selective LRRK2 inhibitors with efficacy in cellular/animal models with good in vivo pharmacokinetics (PK).

In one embodiment the invention includes the compounds of Formula I, II and VII and pharmaceutically acceptable salts thereof as described below.

In one embodiment the present invention includes compounds and compositions of matter including compounds active on protein kinases broadly, including but not limited to ABL1 T315I (SEQ ID NO 1), ALK2 (SEQ ID NO 2), ARK5 (SEQ ID NO 64), Aurora A (SEQ ID NO 3), Aurora B (SEQ ID NO 4), Aurora C (SEQ ID NO 5), BRAF (SEQ ID NO 6), BRAF V599E (SEQ ID NO 7), CHK1 (SEQ ID NO 8), CHK2 (SEQ ID NO 9), C-KIT (SEQ ID NO 10), CLK1 (SEQ ID NO 11), CLK2 (SEQ ID NO 12), CLK3 (SEQ ID NO 13), CLK4 (SEQ ID NO 14), CSF1R (SEQ ID NO 15), DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17), DYRK2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20), FLT3 (SEQ ID NO 21), GSK3β (SEQ ID NO 22), JAK1 (SEQ ID NO 23), JAK2 (SEQ ID NO 24), JAK3 (SEQ ID NO 25), KDR (SEQ ID NO 26), LCK (SEQ ID NO 27), LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), LRRK2 T1602S R1628P T2352M (SEQ ID NO 29), MAP4K4 (SEQ ID NO 30), MELK (SEQ ID NO 31), MLK (SEQ ID NO 32), PDGFR (SEQ ID NO 41), PKA (SEQ ID NO 63), PKCα (SEQ ID NO 42), PKCτ (SEQ ID NO 43), PIM1 (SEQ ID NO 44), RET (SEQ ID NO 45), ROCK1 (SEQ ID NO 46), ROCK2 (SEQ ID NO 47), RSK1 (SEQ ID NO 48), RSK2 (SEQ ID NO 49), p70S6K (SEQ ID NO 50), SGK1 (SEQ ID NO 51), SNF1LK (SEQ ID NO 52), SIK2 (SEQ ID NO 53), SYK (SEQ ID NO 54), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56), TRKB (SEQ ID NO 57) or TRKC (SEQ ID NO 58) kinases and mutations thereof. In another embodiment compounds disclosed herein target two common LRRK2 polymorphisms and Alzheimer's disease progression. The LRRK2 T1602S and T2352M are reported to be greatly associated with the conversion from mild cognitive impairment to AD. The polymorphism of LRRK2 is a suggestive biomarker and LRRK2 modulating agents can be used for the treatment of AD selected patients.

In another embodiment compounds disclosed herein target LRRK2 R1628P variant (SEQ ID NO 29). Dual PD and AD pathologies can co-exist where the LRRK2 R1628P variant (SEQ ID NO 29) is found.

Dual specificity tyrosine (Y) phosphorylation regulated kinase 1A (DYRK1A) (SEQ ID NO 16), a serine/threonine kinase known to play a vital role in neurodevelopment, is activated by autophosphorylation at the Tyr-321 residue. Transgenic mice in which DYRK1A (SEQ ID NO 16) is overexpressed showed hippocampal-dependent learning and memory deficits. Recent reports suggest that DYRK1A (SEQ ID NO 16) may also be involved in the pathological mechanisms of APP and Tau, and may accelerate the formation of amyloid plaques and NFTs which are insoluble deposits of β-amyloid and hyperphosphorylated Tau, respectively, causing the early onset of AD pathogenesis in Down's syndrome (DS). In one embodiment, the compounds of Formula I, II and VII are capable of inhibiting DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17) and DYRK2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20) kinases, benefiting individuals with DS in treating mental retardation and AD.

In another embodiment the invention is directed to methods of making the disclosed compounds and pharmaceutical salts thereof.

In another embodiment methods employing the disclosed compounds of Formula I, II and VII are disclosed for regulating protein kinases wherein said protein kinase includes but is not limited to ABL1T315I (SEQ ID NO 1), ALK2 (SEQ ID NO 2), ARK5 (SEQ ID NO 64), Aurora A (SEQ ID NO 3), Aurora B (SEQ ID NO 4), Aurora C (SEQ ID NO 5), BRAF (SEQ ID NO 6), BRAF V599E (SEQ ID NO 7), CHK1 (SEQ ID NO 8), CHK2 (SEQ ID NO 9), C-KIT (SEQ ID NO 10), CLK1 (SEQ ID NO 11), CLK2 (SEQ ID NO 12), CLK3 (SEQ ID NO 13), CLK4 (SEQ ID NO 14), CSF1R (SEQ ID NO 15), DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17), DYRK2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20), FLT3 (SEQ ID NO 21), GSK3β (SEQ ID NO 22), JAK1 (SEQ ID NO 23), JAK2 (SEQ ID NO 24), JAK3 (SEQ ID NO 25), KDR (SEQ ID NO 26), LCK (SEQ ID NO 27), LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), LRRK2 T1602S R1628P T2352M (SEQ ID NO 29), MAP4K4 (SEQ ID NO 30), MELK (SEQ ID NO 31), MLK (SEQ ID NO 32), PDGFR (SEQ ID NO 41), PKA (SEQ ID NO 63), PKCα (SEQ ID NO 42), PKCτ (SEQ ID NO 43), PIM1 (SEQ ID NO 44), RET (SEQ ID NO 45), ROCK1 (SEQ ID NO 46), ROCK2 (SEQ ID NO 47), RSK1 (SEQ ID NO 48), RSK2 (SEQ ID NO 49), p70S6K (SEQ ID NO 50), SGK1 (SEQ ID NO 51), SNF1LK (SEQ ID NO 52), SIK2 (SEQ ID NO 53), SYK (SEQ ID NO 54), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56), TRKB (SEQ ID NO 57) or TRKC (SEQ ID NO 58) kinases. This set of kinases includes the mutations of these kinases and the use thereof in treating diseases and associated conditions with the regulation of the activity of these kinases.

In another embodiment methods are disclosed for the treatment of pain of neuropathic origin, pain of inflammatory origin, cardiovascular disease, rheumatoid arthritis, osteoarthritis, type 2 diabetes, metabolic syndrome and obesity.

In a further embodiment the invention relates to the novel therapeutic uses of the disclosed compounds involved in the inhibition of protein kinases and the compounds used for treating the diseases associated with the protein kinase activities. They are useful as a drug for the treatment and/or prophylaxis of diseases such as cardiac disease, autoimmune disease, inflammatory disease, central nervous system disease, infectious disease, sepsis, septic shock and the like in mammals (e.g., cat, cattle, dog, horse, goat, monkey, human and the like), for example, ichorrhemia, endotoxin shock, exotoxin shock, heart failure, shock, hypotension, rheumatoid arthritis, arthrosteitis, gastritis, ulcerative colitis, peptic ulcer, stress gastric ulcer, Crohn's disease, autoimmune disease, tissue injury and rejection after organ transplantation, ischemic reperfusion disorder, acute coronary microvascular occlusion, shock vascular occlusion (disseminated intravascular coagulation syndrome (DIC) and the like), ischemic brain disorder, arteriosclerosis, pernicious anemia, Fanconi's anemia, sickle cell anemia, pancreatitis, nephrotic syndrome, nephritis, renal failure, insulin-dependent diabetes mellitus, non-insulin dependent diabetes mellitus, hepatic porphyria, alcoholism, Parkinson's disease, chronic leukemia, acute leukemia, tumor, myeloma, relief of side effects of anticancer agent, infant and adult respiratory distress syndrome, pulmonary emphysema, dementia, Alzheimer's disease, multiple sclerosis, vitamin E deficiency, aging, sunburn, myodystrophy, myocarditis, cardiac myopathy, myocardial infarction, sequela of myocardial infarction, osteoporosis, pneumonia, hepatitis, psoriasis, pain, cataract, influenza infection, malaria, human immunodeficiency virus (HIV) infection, radiation injury, burn, improved efficiency of in vitro fertilization, hyperkalaemia, ankylosing spondylitis, osteopenia, bone Behcet's disease, osteomalacia, bone fracture, acute bacterial meningitis, Helicobacter pylori infection, invasive staphylococcal infection, tuberculosis, systemic mycotic infection, herpes simplex virus infection, varicella-zoster virus infection, human papilloma virus infection, acute virus encephalitis, encephalitis, asthma, atopic dermatitis, allergic rhinitis, reflux esophagitis, fever, hypercholesterolemia, hyperglyceridemia, hyperlipidemia, diabetic complications, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, gout, gastric atony, hemorrhoid, systemic lupus erythematodes, spinal trauma, agrypnia, schizophrenia, epilepsy, cirrhosis, hepatic failure, unstable angina pectoris, valvular disease of heart, thrombocytopenia derived from dialysis, acute ischemic stroke, acute brain thrombosis, cancer metastasis, bladder cancer, breast cancer, cervical cancer, colon cancer, stomach cancer, ovarian cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, Hodgkin's disease, non-Hodgkin's lymphoma and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D are graphical depictions of results of testing of selected compounds in accordance with the present disclosure in 10-dose IC₅₀ mode with 3 fold serial dilution starting at 100 μM. The control compound Staurosporine was tested in 10 dose IC50 with 3 fold serial dilution starting at 20 μM;

FIG. 2 is a graphical depiction of mean plasma concentrations of test compound in mice dosed 10 mg/kg PO and 5 mg/kg IV in accordance with one aspect of the invention;

FIG. 3 is a listing including the Leucine-Rich Repeat Kinase (LRRK2)(SEQ ID NO 62) domain region sequence (1879-2140) from the full length protein sequence NP_(—)940980;

FIG. 4 depicts a structure-based sequence alignment in Clustal W of the catalytic protein kinase domains of LRRK2 Mixed Lineage Kinase 1 (MLK1) (SEQ ID NO 33), Transforming growth factor-beta (TGF-β)-activated Kinase 1 (TAK1) (SEQ ID NO 59), BRAF, Tie2 (SEQ ID NO 60) and Type I TGF-β receptor (TβR-I) (SEQ ID NO 61). Amino acid residue annotation were identical residues (*), highly conserved residues (:), and similar residues (.). The active site residues highlighted in yellow and the gatekeeper residues in turquoise and the DFG/DYG residues shown in gray and blue color a G2019S mutant site; and

FIG. 5 is a graphical depiction of A) Homology model of LRRK2 (SEQ ID NO 62) in complex with one of the lead inhibitors 13; B) The critical active site residues shown in color-by-atom in stick representations; C) The LRRK2 active site depicted in surface in complex with one of the lead series compounds belongs to 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine structural class in accordance with at least one aspect of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID No. 1 is the homo sapiens (human) ABL T315I protein kinase sequence;

SEQ ID No. 2 is the homo sapiens ALK2 protein kinase sequence;

SEQ ID No. 3 is the human Aurora A protein kinase sequence;

SEQ ID No. 4 is the human Aurora B protein kinase sequence;

SEQ ID No. 5 is the human Aurora C protein kinase sequence;

SEQ ID No. 6 is the human BRAF protein kinase sequence;

SEQ ID No. 7 is the human BRAF V599E protein kinase sequence;

SEQ ID No. 8 is the human CHK1 protein kinase sequence;

SEQ ID No. 9 is the human CHK2 protein kinase sequence;

SEQ ID No. 10 is the human C-KIT protein kinase sequence;

SEQ ID No. 11 is the human CLK1 protein kinase sequence;

SEQ ID No. 12 is the human CLK2 protein kinase sequence;

SEQ ID No. 13 is the human CLK3 protein kinase sequence;

SEQ ID No. 14 is the human CLK4 protein kinase sequence;

SEQ ID No. 15 is the human CSF1R protein kinase sequence;

SEQ ID No. 16 is the human DYRK1A protein kinase sequence;

SEQ ID No. 17 is the human DYRK1B protein kinase sequence;

SEQ ID No. 18 is the human DYRK2 protein kinase sequence;

SEQ ID No. 19 is the human DYRK3 protein kinase sequence

SEQ ID No. 20 is the human DYRK4 protein kinase sequence;

SEQ ID No. 21 is the human FLT3 protein kinase sequence;

SEQ ID No. 22 is the human GSK3β protein kinase sequence;

SEQ ID No. 23 is the human JAK1 protein kinase sequence;

SEQ ID No. 24 is the human JAK2 protein kinase sequence;

SEQ ID No. 25 is the human JAK3 protein kinase sequence;

SEQ ID No. 26 is the human KDR protein kinase sequence;

SEQ ID No. 27 is the human LCK protein kinase sequence;

SEQ ID No. 28 is the human LRRK2 G2019S protein kinase sequence;

SEQ ID No. 29 is the human LRRK2 T1602S R 1628P T2352M protein kinase sequence;

SEQ ID No. 30 is the human MAP4K4 protein kinase sequence;

SEQ ID No. 31 is the human MELK protein kinase sequence;

SEQ ID No. 32 is the human MLK protein kinase sequence;

SEQ ID No. 33 is the human MLK1 protein kinase sequence;

SEQ ID No. 34 is the human MAP3K9 protein kinase sequence;

SEQ ID No. 35 is the human MAP4K5 protein kinase sequence;

SEQ ID No. 36 is the human MINK1 protein kinase sequence;

SEQ ID No. 37 is the human MARK1 protein kinase sequence;

SEQ ID No. 38 is the human MARK2 protein kinase sequence;

SEQ ID No. 39 is the human MARK3 protein kinase sequence;

SEQ ID No. 40 is the human MARK4 protein kinase sequence;

SEQ ID No. 41 is the human PDGFR protein kinase sequence;

SEQ ID No. 42 is the human PKCα protein kinase sequence;

SEQ ID No. 43 is the human PKCτ protein kinase sequence;

SEQ ID No. 44 is the human PIM1 protein kinase sequence;

SEQ ID No. 45 is the human RET protein kinase sequence;

SEQ ID No. 46 is the human ROCK1 protein kinase sequence;

SEQ ID No. 47 is the human ROCK2 protein kinase sequence;

SEQ ID No. 48 is the human RSK1 protein kinase sequence;

SEQ ID No. 49 is the human RSK2 protein kinase sequence;

SEQ ID No. 50 is the human p70S6K protein kinase sequence;

SEQ ID No. 51 is the human SGK1 protein kinase sequence;

SEQ ID No. 52 is the human SNF1LK protein kinase sequence;

SEQ ID No. 53 is the human SIK2 protein kinase sequence;

SEQ ID No. 54 is the human SYK protein kinase sequence;

SEQ ID No. 55 is the human TNIK protein kinase sequence;

SEQ ID No. 56 is the human TRKA protein kinase sequence;

SEQ ID No. 57 is the human TRKB protein kinase sequence;

SEQ ID No. 58 is the human TRKC protein kinase sequence;

SEQ ID No. 59 is the human TAK1 protein kinase sequence;

SEQ ID No. 60 is the human Tie2 protein kinase sequence;

SEQ ID No. 61 is the human TGFβ1 ALK5 protein kinase sequence;

SEQ ID No. 62 is the human LRRK2 protein kinase sequence;

SEQ ID No. 63 is the human PKA protein kinase sequence; and

SEQ ID No. 64 is the human ARK5 protein kinase sequence.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the subject matter provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

In one aspect the invention provides compounds having the chemical structure of Formula I and II:

and pharmaceutically acceptable salts, isomers and tautomers thereof, wherein: in Formula I, X is independently —N or —CH; L₁ is independently a direct bond, substituted or unsubstituted 6-membered aryl, heteroaryl, substituted or unsubstituted 5-membered aryl or heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, —C(O)_(n), S(O)n, —O, —NH, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted 2 to 5 membered heteroalkylene groups; where “n” is from 0 to 2, hydrogen, —CH₃, halo, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, —OR₄ —SR₄, —NHR₄, —C(O)R₄—, —C(S)R₄, —C(O)NR₄, and —S(O)₂R₄, —S(O)₂NHR₄, —NHCOR₄, —CONHR₄, CONR₄, —CH═CHR₄, —NCH₃R₄, R₄ is optionally substituted at each occurrence and is selected from alkyl, heteroaryl, cycloalkyl, heterocycloalkyl, —C₁₋₆ alkyl, —C₁₋₄ haloaliphatic, —C₂₋₆ alkenyl, mono/dialkylamino, aryl C₄₋₇ heterocycloakyl, —C₅₋₆ aryl and, wherein the functional groups are each optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted lower alkyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino functional groups or additional substituents selected from —NR₅R₆ and —CR₅R₆ respectively, and wherein; R₄ together with the carbon to which it is attached may form a 3-7 member monocyclic cycloalkyl, 5-6 membered monocyclic heterocycloalkyl, heterocycloalkyl, heteroaryl; wherein the monocyclic cycloalkyl or heterocycloalkyl are each independently optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted lower alkyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino; R₅ and R₆ are each independently —H, cycloalkyl, alkyl, heterocycloalkyl, aryl, heteroaryl, —C₁₋₆ alkyl, —C₃₋₇ cycloalkyl, —C₃₋₆ heterocyclocycloalkyl, each optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino; wherein L1 is optionally substituted with —R₇ or Y₉; wherein R₇ is independently a bond, substituted or unsubstituted —C═S, C═O, —C₁₋₆ alkyl, each optionally further substituted by C₁₋₆ alkyl, (for example, (—CH₂)_(n), wherein n is an integer from 0-3), —O, —S, —SO, SO₂, —NH or —CH═CH groups; and R₇ may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, wherein C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl are each optionally substituted with one or more of fluoro, —OH, —NH₂, OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, mono/dialkylamino, and alkyl carbon bound to the N of —C(O)NHR₈, —C(S)NHR₈ or —S(O)₂NHR₈; wherein the alkyl chain(s) of OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, and mono/dialkylamino are each optionally independently substituted with one or more of fluoro, —OH, —NH₂, wherein any substitution of the alkyl chain carbon bound to O of alkoxy, S of thioalkyl or N of mono/dialkylamino is fluoro; R₈ combines with the nitrogen to which it is attached to form a 5-7 member heterocycloalkyl optionally substituted with one or more of fluoro, —OH, —NH2, C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, fluoro substituted C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, OCH₃, CH₂OCH₃, fluoro substituted OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, and fluoro-substituted methylthio, ethylthio, butylthio, isobutylthio; Y₉ is —C₁₋₆ alkyl, C₃₋₇ cycloalkyl, —C₁₋₆-alkyl, —C₃₋₇-cycloalkyl, —C₁₋₆ alkyl heteroaryl, —C₄₋₇ heterocycloalkyl, aryl or heteroaryl, each optionally independently substituted with one or more substituents selected from R₄; and R₁, R₂ and R₃ are each independently —H, halo, —CN, —NH₂, —C₁₋₆ aliphatic, —OC₁₋₆ aliphatic, —C₃₋₆ cycloaliphatic, —OC₃₋₆ cycloaliphatic, —NH₂, —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, —CONH₁₋₆ heterocycloalkyl, —NHC₁₋₆ aliphatic, —NHC₃₋₆ cycloaliphatic, —NHS(O)₂C₁₋₆ aliphatic, —NHS(O)₂C₃₋₆ cycloaliphatic, —NHS(O)₂ phenyl, —NHS(O)₂ benzyl, —NHS(O)₂ heteroaryl, —S(O)₂C₁₋₆ aliphatic, —S(O)₂C₃₋₆ cycloaliphatic, —S(O)₂ phenyl, —S(O)₂ benzyl, —S(O)₂ heteroaryl, —S(O)₂NHC₁₋₆ aliphatic, —S(O)₂NHC₃₋₆ cycloaliphatic, —S(O)₂NHphenyl, —S(O)₂NHbenzyl, or —S(O)₂NHheteroaryl, wherein each of said heteroaryl of R₁, R₂ and R₃ is independently a 5- or 6-member ring having 1, 2, or 3 atoms selected from N, O, or S, and wherein said aliphatic, cycloaliphatic, phenyl, benzyl, or heteroaryl of R₁, R₂ and R₃ is each optionally substituted with 1, 2, or 3 ZR₁₀ groups; wherein ZR₁₀ is halo, oxo, —C₁₋₂ alkyl, substituted C₁₋₂ alkyl with 1-3 —F or —Cl atoms, —C₃₋₆ cycloalkyl, —OH, —OC₁₋₂ alkyl, —OC₁₋₂ alkyl substituted with 1-3 —F or Cl atoms, —C(O)C₁₋₂ alkyl, or —SC₁₋₂ alkyl.

In one embodiment, the invention relates to a compound having a structure represented by a Formula I where L₁ is more specifically;

indicates the attachment to L₁ of Formula I; wherein R is —H, halo, —CH₃, —CH₂CH₃, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, OH, —CH₂OH, —SO₂CH₃, SO₂N(CH₃)₂, —COCH₃; and wherein “n” is an integer from 0 to 2.

In one embodiment R₁, R₂ and R₃ are each independently:

wherein

indicates the attachment to R₁, R₂ and R₃ of Formula I;

In another embodiment R₁, R₂ and R₃ at each occurrence is independently —H, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

Lower alkyl as used herein means and includes C₁₋₆ alkyl including cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In a further embodiment R₁, R₂ and R₃ are —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, or —CONH₁₋₆ heterocycloalkyl.

In one embodiment R₁, R₂ and R₃ are each independently directly substituted by —H, —F, —Cl, —OCF₃, CF₃, —CH₃, —OCHF₂ or —OCH₃, wherein at least one of R₁ R₂ and R₃ is not hydrogen.

In a further embodiment R₁, R₂ and R₃ are each independently formula III and IV

wherein

indicates the attachment to Formula I of one of R₁, R₂ and R₃ positions and R₉ is —H, —CH₃, —CH(CH₃)₂, —CH₂CH₃, —CH₂CH₂—OH, or —C3-6-alkyl.

In another embodiment, the invention relates to compound of formula VIII wherein

indicates attachment to R₁, R₂ and R₃ of Formula I respectively

wherein R₁₂ is —H, halo, —CH₃, —CH₂CH₃, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, OH, —CH₂OH, —SO₂CH₃, SO₂N(CH₃)₂, or —COCH₃.

In another aspect, in Formula II,

Q is —NH, —O, —S, —NCH₃, —NHCO, —CONH, a direct bond, substituted or unsubstituted 6-membered aryl, heteroaryl, substituted or unsubstituted 5-membered aryl or heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, —C(O)_(n), S(O)n, —O, —NH, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted 2 to 5 membered heteroalkylene groups, where “n” is an integer from 0 to 2, or one of

wherein

indicates the attachment to Q of Formula II; L₁ is as described above for Formula I; L₂ is a Formula V

wherein X₁ is —N, —CH; X₂ is —N, —CH and X₃ is —N, —CH respectively

indicates the attachment to L₂ of Formula II; R₁₀ is —H, halo, —CN, —NH₂, —C₁₋₆ aliphatic, —OC₁₋₆ aliphatic, —C₃₋₆ cycloaliphatic, —OC₃₋₆ cycloaliphatic, —NH₂, —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, —CONH₁₋₆ heterocycloalkyl, —NHC₁₋₆ aliphatic, —NHC₃₋₆ cycloaliphatic, —NHS(O)₂C₁₋₆ aliphatic, —NHS(O)₂C₃₋₆ cycloaliphatic, —NHS(O)₂ phenyl, —NHS(O)₂ benzyl, —NHS(O)₂ heteroaryl, —S(O)₂C₁₋₆ aliphatic, —S(O)₂C₃₋₆ cycloaliphatic, —S(O)₂ phenyl, —S(O)₂ benzyl, —S(O)₂ heteroaryl, —S(O)₂NHC₁₋₆ aliphatic, —S(O)₂NHC₃₋₆ cycloaliphatic, —S(O)₂NH phenyl, —S(O)₂NH benzyl, or —S(O)₂NH heteroaryl, wherein said heteroaryl of R₁₀ is a 5- or 6 member ring having 1, 2, or 3 atoms selected from N, O, or S, and wherein said aliphatic, cycloaliphatic, phenyl, benzyl, or heteroaryl of R₁₀ are each independently optionally substituted with 1, 2, or 3 ZR₁₀ groups; wherein ZR₁₀ is halo, oxo, —C₁₋₂ alkyl, substituted C₁₋₂ alkyl with 1-3 —F, —Cl atoms, —C₃₋₆ cycloalkyl, —OH, —OC₁₋₂ alkyl, —OC₁₋₂ alkyl substituted with 1-3 —F, —Cl atoms, —C(O)C₁₋₂ alkyl, or —SC₁₋₂ alkyl.

In one embodiment ZR₁₀ is directly substituted by —H, —F, —Cl, —OCF₃, CF₃, —CH₃, —OCHF₂ or —OCH₃, wherein at least one of R₁₀ is not hydrogen;

In yet another embodiment, the invention comprises a compound of Formula II where R₁₀ is

wherein

indicates the attachment to R₁₀ of Formula V; X₃ and X₄ are each independently either —N or —CH; Q₁ is a bond, —CH₂, or —CH₂ and (n1) is 0, 1 or 2; and R₁₁ is optionally substituted aryl, —C₅₋₆ aryl, —C₅₋₆ heteroaryl optionally substituted with one or more substituent —H, —CH₃, halo, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, OH.

In another embodiment, R₁₀ is formula III or IV.

In another aspect, the invention relates to a compound of formula VII

wherein X is independently —N or —CH, and R₁, R₂, R₃ and L₁ are as described in all embodiments above for Formula I.

The presently disclosed subject matter invention relates to the substituted 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine and pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors. The invention is directed in one embodiment to pharmaceutical compositions containing the Formula I, II and VII compounds and methods of using the compounds, pharmaceutically acceptable salts thereof and/or compositions thereof to treat various types of diseases or conditions such as Parkinson's disease, Alzheimer's disease, Down's syndrome, Huntington's disease, other neurodegenerative diseases and or CNS diseases, cancer, metabolic and inflammatory disease.

In another embodiment methods are disclosed for the treatment of pain of neuropathic origin, pain of inflammatory origin, cardiovascular disease, rheumatoid arthritis, osteoarthritis, type 2 diabetes, metabolic syndrome and obesity.

The invention is also directed to methods of making the disclosed compounds and pharmaceuticals salts thereof.

In another embodiment methods employing the disclosed compounds of Formulae I, II and VII are disclosed for regulating protein kinases wherein said protein kinase i includes but is not limited to ABL1T315I (SEQ ID NO 1), ALK2 (SEQ ID NO 2), ARK5 (SEQ ID NO 64), Aurora A (SEQ ID NO 3), Aurora B (SEQ ID NO 4), Aurora C (SEQ ID NO 5), BRAF (SEQ ID NO 6), BRAF V599E (SEQ ID NO 7), CHK1 (SEQ ID NO 8), CHK2 (SEQ ID NO 9), C-KIT (SEQ ID NO 10), CLK1 (SEQ ID NO 11), CLK2 (SEQ ID NO 12), CLK3 (SEQ ID NO 13), CLK4 (SEQ ID NO 14), CSF1R (SEQ ID NO 15), DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17), DYRK2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20), FLT3 (SEQ ID NO 21), GSK3β (SEQ ID NO 22), JAK1 (SEQ ID NO 23), JAK2 (SEQ ID NO 24), JAK3 (SEQ ID NO 25), KDR (SEQ ID NO 26), LCK (SEQ ID NO 27), LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), LRRK2 T1602S R1628P T2352M (SEQ ID NO 29), MAP4K4 (SEQ ID NO 30), MELK (SEQ ID NO 31), MLK (SEQ ID NO 32), PDGFR (SEQ ID NO 41), PKA (SEQ ID NO 63), PKCα (SEQ ID NO 42), PKCτ (SEQ ID NO 43), PIM1 (SEQ ID NO 44), RET (SEQ ID NO 45), ROCK1 (SEQ ID NO 46), ROCK2 (SEQ ID NO 47), RSK1 (SEQ ID NO 48), RSK2 (SEQ ID NO 49), p70S6K (SEQ ID NO 50), SGK1 (SEQ ID NO 51), SNF1LK (SEQ ID NO 52), SIK2 (SEQ ID NO 53), SYK (SEQ ID NO 54), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56), TRKB (SEQ ID NO 57) or TRKC (SEQ ID NO 58) kinases. This set of kinases includes the mutations of these kinases and the use thereof in treating diseases and associated conditions with the regulation of the activity of these kinases.

In a further embodiment the invention relates to the novel therapeutic use of the disclosed compounds involved in the inhibition of protein kinases and the compounds used for treating the diseases associated with the protein kinase activities. They are useful as a drug for the treatment and/or prophylaxis of diseases such as cardiac disease, autoimmune disease, inflammatory disease, central nervous system disease, infectious disease, sepsis, septic shock and the like in mammals (e.g., cat, cattle, dog, horse, goat, monkey, human and the like), for example, ichorrhemia, endotoxin shock, exotoxin shock, heart failure, shock, hypotension, rheumatoid arthritis, arthrosteitis, gastritis, ulcerative colitis, peptic ulcer, stress gastric ulcer, Crohn's disease, autoimmune disease, tissue injury and rejection after organ transplantation, ischemic reperfusion disorder, acute coronary microvascular occlusion, shock vascular occlusion (disseminated intravascular coagulation syndrome (DIC) and the like), ischemic brain disorder, arteriosclerosis, pernicious anemia, Fanconi's anemia, sickle cell anemia, pancreatitis, nephrotic syndrome, nephritis, renal failure, insulin-dependent diabetes mellitus, non-insulin dependent diabetes mellitus, hepatic porphyria, alcoholism, Parkinson's disease, chronic leukemia, acute leukemia, tumor, myeloma, relief of side effects of anticancer agent, infant and adult respiratory distress syndrome, pulmonary emphysema, dementia, Alzheimer's disease, multiple sclerosis, vitamin E deficiency, aging, sunburn, myodystrophy, myocarditis, cardiac myopathy, myocardial infarction, sequela of myocardial infarction, osteoporosis, pneumonia, hepatitis, psoriasis, pain, cataract, influenza infection, malaria, human immunodeficiency virus (HIV) infection, radiation injury, burn, improved efficiency of in vitro fertilization, hypercalcemia, ankylosing spondylitis, osteopenia, bone Behcet's disease, osteomalacia, bone fracture, acute bacterial meningitis, Helicobacter pylori infection, invasive staphylococcal infection, tuberculosis, systemic mycotic infection, herpes simplex virus infection, varicella-zoster virus infection, human papilloma virus infection, acute virus encephalitis, encephalitis, asthma, atopic dermatitis, allergic rhinitis, reflux esophagitis, fever, hypercholesterolemia, hyperglyceridemia, hyperlipidemia, diabetic complications, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, gout, gastric atony, hemorrhoid, systemic lupus erythematodes, spinal trauma, agrypnia, schizophrenia, epilepsy, cirrhosis, hepatic failure, unstable angina pectoris, valvular disease of heart, thrombocytopenia derived from dialysis, acute ischemic stroke, acute brain thrombosis, cancer metastasis, bladder cancer, breast cancer, cervical cancer, colon cancer, stomach cancer, ovarian cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, Hodgkin's disease, non-Hodgkin's lymphoma and the like.

Methods of Preparation of Compounds EXAMPLES

In certain embodiments, the Examples depicted below are compounds prepared according to general procedures given in the following sections. Although the synthetic methods and Schemes depict the syntheses of certain compounds of the present invention, the methods and other methods known to one of ordinary skill in the art can be applied to all the compounds of the genus, the genus sub-class and species of each of these compounds as described herein.

All aspects of this invention can be understood from the following Schemes. The following are exemplary and are not intended to limit the scope of the invention.

The synthesis of 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine and pyrazolo[3,4-b]pyridine derivatives claimed in formula I and II will be apparent to the skilled artisan based on the methods of synthesis provided in the general Schemes 1 and 2. Some of these synthetic methods described here in the Examples section below may be employed and or expanded to obtain the compounds of Formula I and II.

L₂, R₂, R₃, R₁₃ and ( )_(n1) are defined above. “PG” represents a protecting group, or protecting groups (PGs), and refers to a group used to protect a certain functional moiety to prevent it from participation in chemical reactions until the PG is removed. As is well known to the skilled artisan, these protective groups can be removed by acid, base, and hydrogenolysis conditions in the presence or absence of organic solvents. Such PGs employed in the above synthesis schemes include, but are not limited to p-methoxybenzyl (PMB), 2,4-dimethoxybenzyl, benzyl (Bn), 4-toluenesulfonyl chloride (tosyl chloride or TsCl), 2-(trimethylsilyl) ethoxymethyl chloride (SEM-Cl), trityl chloride (triphenylmethyl chloride), dimethoxytrityl, tetrahydropyranyl (THP), di-tert-butyl dicarbonate (tert-Boc), fluorenylmethyloxycarbonyl chloride (FMOC-Cl), tert-butyldimethylsilyl chloride (TBDMS-Cl), and carboxybenzyl (Cbz) groups.

Experimental Details and Examples

Melting points were determined in a MP-96 digital Polmon apparatus. ¹H NMR and ¹³C NMR spectra were recorded at room temperature in CDCl3 or DMSO-d6 at Jeol 400-MHz NMR spectrophotometer using solvent peaks for CDCl3: 7.27 and DMSO-d6 2.50 (D) as internal references. The assignment of chemical shifts is based on standard NMR experiments (1H, 13C). Mass spectra were recorded on a Shimadzu LCMS LC-210EV spectrometer with an API-ES ionization source. Jasco-FTIR-4100 was used to record the IR spectra. TLC analyses were performed on silica F254 and detection by UV light at 254 nm, or by spraying with phosphomolybdic-H₂SO₄ dyeing reagent, KMNO₄ or iodine. Column chromatography were performed on silica Gel 60 (230 mesh). Purifications and separations were performed on a standard silica flash chromatography system. The purity of the samples has been determined by HPLC for the % area peak corresponding to the retention of compound and elemental analysis for C, H, N and O was carried out using Perkin-Elmer 2400 elemental analyser and chloride analysis performed using calorimetric titration at the Intertek USA Inc., QTI.

Abbreviations

-   CH₂Cl₂ or DCM Dichloromethane -   CHCl3 Chloroform -   DMF N,N-Dimethylformamide -   DME 1,2-Dimethoxyethane -   DMSO Dimethylsulfoxide -   THF Tetrahydrofuran -   MeOH Methanol -   EtOH Ethanol -   EtOAc Ethylacetate -   AcCN/MeCN Acetonitrile -   DIPEA Diisopropylethylamine -   IP Isopropanol -   Na₂CO₃ Sodium Carbonate -   K₂CO₃ Potassium Carbonate -   Cs₂CO₃ Cesium Carbonate -   TFA Trifluoroacetic acid -   RM Reaction Mixture -   RT Room Temperature -   SM Starting Material -   TLC Thin Layer Chromatography -   HPLC High-Performance Liquid Chromatography -   NMR Nuclear Magnetic Resonance Spectrometer -   MS Mass Spectrometer -   LCMS Liquid Chromatography Mass Spectrometer -   UV Ultra Violet Spectrometer -   PG Protecting Group -   DMSO-d₆ Dimethylsulfoxide deuterated solvent -   CDCl₃ Chloroform deuterated solvent -   Pd(PPh₃)₄ Tetrakis(triphenylphosphine)palladium(0) -   Pd₂(dba)₃ Tris(dibenzylideneacetone)dipalladium(0) -   Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene -   ±BINAP rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl -   Pd(dppf)Cl₂.CH₂Cl₂     [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) DCM -   EDC HCl N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide HCl -   HOBT 1-Hydroxybenzotriazole hydrate

Example 1 Preparation of Sodium Nitromalonaldehyde

A stirred solution of mucobromic acid (51 g) in water (50 ml) was heated to 55° C., and then to it a hot solution of sodium nitrite in ethanol (50 ml) was added in about 45 minutes. The RM was then stirred at 50° C. for another 15 minutes. Then the RM was cooled to 0-5° C. and the reaction mixture was filtered. The wet cake was taken into 100 ml of EtOH and 25 ml of water heated to reflux for 15 minutes. The compound was filtered in hot condition. The unwanted yellow residue solid was discarded. The filtrate was cooled to 0° C.; solid was precipitated, the solid was filtered and dried under vacuum to afford the product as yellow solid, 8 g (Yield-26%) and was confirmed as the desired product 2, by IR and MR.

Example 2 3-Methyl-5-nitro-1H-pyrazole-[3,4-b]pyridine

To a stirred solution of 3-Methyl-1H-pyrazole-5-amine (14 g, 144.15 mmol) in acetic acid (193.05 ml) and water (20 ml), sodium nitro-malonaldehyde (25 g, 158.56 mmol) was added at room temperature. The reaction mixture was heated to 100° C. for 12 h. TLC was checked. Then the reaction mass was cooled to RT and poured into water (200 ml). The precipitate was filtered and washed with water (200 ml) followed by diethyl ether (300 ml). It was dried to afford compound 4 as pale yellow solid; 14 g (Yield-55%). The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, DMSO-d₆) δ: 14.1 (bs, 1H), 9.3 (s, 1H), 9.2 (s, 1H), 2.55 (s, 3H); (MS=178, [M+1]).

Example 3 3-Methyl-1H-pyrazolo[3,4-b]pyridin-5-amine

To a stirred solution 3-Methyl-5-nitro-1H-pyrazole[3,4-b]pyridine (14 g, 78.65 mmol) in ethanol, Pd/C (4 g) was added and the reaction mixture was stirred at RT for 5 h. Reaction mixture was filtered through a Celite bed. All volatiles were removed under reduced pressure to get the desired product as brown solid, 9 g (Yield-80%). The product 5 was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.62 (bs, 1H), 8 (s, 1H), 7.89 (s, 1H), 5.93 (s, 1H), 2.38 (s, 1H); MS=148, (M+1).

Example 4 5-Bromo-3-methyl-1H-pyrazolo-[3,4-b]pyridine

A stirred solution 3-Methyl-1H-pyrazole[3,4-b]pyridin-5-amine (200 mg, 1.35 mmol) in 48% hydrobromic acid (1.46 ml, 27.02 mmol) and water (1.5 ml) was cooled to −5° C. A solution of sodium nitrite (100 mg, 1.47 mmol) in water (0.5 ml) was added to this reaction mixture at 0° C. Then this reaction mixture was added to a suspension of copper (I) bromide (300 mg, 2.02 mmol) in 48% hydrobromic acid (0.4 ml) maintained at 0° C. Reaction mixture was stirred for 4 h at RT. Reaction mixture was extracted with ethyl acetate. The extract was washed with water (3×20 ml) and sodium carbonate solution (2×15 ml). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column over 230-400 mesh silica gel eluting with 25% ethyl acetate/petroleum ether to get the desired product 6, 100 mg (Yield-35%) as white solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 10.92 (bs, 1H), 8.59 (s, 1H), 8.21 (s, 1H), 2.59 (s, 1H); MS: 212 (M+1).

Example 5 5-Bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 5-Bromo-3-methyl-1H-pyrazolo[3,4-b]pyridine (1.3 g, 6.13 mmol) in DMF, sodium hydride (0.49 g, 12.24 mmol) was added portion wise at −5° C., then the reaction mixture was stirred at RT for 1 h. It was again cooled to 0° C., and 4-methoxybenzyl chloride slowly added and stirred overnight at RT. Reaction mixture was concentrated and diluted with water and extracted with ethyl acetate (3×50 ml). The combined extract was washed with water, dried over anhydrous sodium sulphate, filtered and concentrated. The product was purified by flash column chromatography over 230-400 mesh silica gel eluting with 20% ethyl acetate/petroleum ether to afford the desired product 7, 1 g (Yield: 55%) as white solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 8.58 (s, 1H), 8.15 (s, 1H), 7.3 (d, 2H), 6.81 (d, 2H), 5.57 (s, 2H), 3.78 (s, 3H), 2.53 (s, 3H); MS: 333, (M+1), LCMS˜87%.

Example 6 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 5-Bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (1 g, 3.003 mmol) in DMF, potassium acetate (883 mg, 19.009 mmol), followed by bis-(pinacolato-diborane) (1.5 g, 6.006 mmol) was added. The reaction mixture was degassed with argon for 30 min. Then bis-(triphenylphosphine)-palladium (II) chloride (210 mg, 0.30 mmol), was added and again degassed with argon for 30 minutes. Then RM was heated to 90° C., stirred overnight. Reaction mixture was concentrated under reduced pressure and the obtained residue was diluted with water and extracted with ethyl acetate (3×50 ml). The combined extract was washed with water, dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column using 230-400 mesh silica gel and 20% ethyl acetate/petroleum ether to afford the desired product 8, 850 mg (Yield: 80%) as white solid. The product was confirmed by ¹HNMR and LCMS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 1.38 (s, 9H), 2.55 (s, 3H), 3.77 (s, 3H), 5.59 (s, 2H), 6.8 (d, 2H), 7.29 (d, 2H); MS: 379 (M+1), LCMS˜94%.

Example 7 6-bromo-N-(1-methylpiperidine4-yl)pyrazine-2-amine

To a stirred solution of 2-bromo-6-chloro pyridine (500 mg, 2.58 mmol) in DMF, DIPEA was added followed by 4-amino-N-methylpiperidine and was heated at 80° C. overnight. Reaction mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate (50 ml), the extract was washed with water. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column using 230-400 mesh silica gel eluting with 10% MeOH/DCM system to afford the desired product 11, 95 mg (Yield: 14%) as pale yellow solid. LCMS showed it as a mixture of Br/Cl isomers and so ¹HNMR was not clean, but characteristic peaks were present.

Example 8 6-(1-(4-methoxybenzyl)-3methyl-1H-pyrazolo[3,4-b]pyridine-5yl)-N-(1-methyl piperidine-4yl)pyrazin-2-amine

To a stirred solution of 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (320 mg, 0.841 mmol) in EtOH (0.5 ml), water (0.5 ml) and DME, 6-bromo-N-(1-methylpiperidine4-yl)pyrazine-2-amine (190 mg, 0.725 mmol) and sodium carbonate (163 mg, 1.54 mmol) were added. Then the reaction mixture was degassed with argon for 30 min. Tetrakis(triphenylphosphine)palladium (81 mg, 0.841 mmol) was added to this reaction mixture, which was degassed with argon for another 10 min. Then it was heated at 90° C. overnight. Reaction mixture was concentrated and diluted with water and extracted with ethyl acetate. The extract was dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column using 230-400 mesh silica gel eluting with 10% MeOH/DCM system to afford the desired product 12, 35 mg (Yield: 10%) as dark brown solid. The product was confirmed by ¹HNMR (not clean but characteristic peaks were present) and LCMS (92%) spectrum analysis.

Example 9 6-(3methyl-1H-pyrazolo[3,4-b]pyridine-5yl)-N-(1-methylpiperidine-4yl)pyrazin-2-amine

A stirred solution of 1-(4-methoxybenzyl)-3methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (35 mg) in DCM (1 ml) was cooled to 0° C. Then TFA (4 ml) was added to it and the mixture was stirred overnight at 60° C. TLC was checked. Reaction mixture was concentrated and basified with saturated sodium bicarbonate solution, and again concentrated. The crude mass was purified by flash column chromatography over basic alumina using 1-3% MeOH/DCM system to afford the desired product 13 as pale yellow solid, 14 mg (Yield: 56%). The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 11.85 (bs, 1H), 9.2 (s, 1H), 8.59 (s, 1H), 8.38 (s, 1H), 7.86 (s, 1H), 7.26 (s, 1H), 4.77 (d, 1H), 3.85 (bs, 1H), 2.91 (d, 2H), 2.67 (s, 3H), 2.36 (s, 3H), 2.32 (t, 2H), 2.15 (d, 2H), 1.66 (m, 2H); MS: 324.10 (M+1), LCMS˜98.99%.

Example 10 6-chloro-N-(1-methylpiperidin-4-yl)pyrazin-2-amine

To a stirred solution of 2-bromo-6-chloro pyridine (500 mg, 2.58 mmol) in DMF, DIPEA was added followed by 4-amino-N-methylpiperidine and was heated at 80° C. overnight. Reaction mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate (50 ml), the extract was washed with water. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column using 230-400 mesh silica gel eluting with 10% MeOH/DCM system to afford the desired product 11, 95 mg (Yield: 14%) as pale yellow solid. LCMS confirms the mixture of Br/Cl isomers, majorly compound 11. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.41 (s, 1H), 7.95 (s, 1H), 3.70 (s, 4H), 2.89 (s, 2H), 2.73 (bs, 2H), 2.01 (s, 1H), 1.98 (bs, 3H); MS: 226.8 (M+1).

Example 11 5-bromopyrimidin-4-ol

To a stirred solution pyridine-4-ol (500 mg, 5.208 mmol) in acetic acid maintained at 4-6° C., bromine was slowly added. The cooling mixture was removed and stirred at RT for 3 h. TLC was checked. Reaction mixture was concentrated and the residue was washed with ether to get the desired product 14, as brown solid, 650 mg (Yield-72%). The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 9.45-9.7 (bs, 3H), 8.29 (s, 1H), 8.23 (s, 1H); MS-174 (M+1).

Example 12 5-bromo-4-chloropyrimidine

A stirred solution of 5-bromopyrimidin-4-ol (650 mg, 3.73 mmol) in POCl₃ (in excess) was heated at 100° C. overnight. The reaction mixture was cooled and concentrated under reduced pressure. Crude product was dissolved in ethyl acetate and this mixture was slowly poured into saturated sodium bicarbonate solution. The ethyl acetate layer was separated and washed with brine solution, and dried over anhydrous sodium sulphate and concentrated to get the desired product 15 as yellow solid, 300 mg (Yield-43%). The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 8.86 (s, 1H), 8.82 (s, 1H); MS-192 (M+1).

Example 13 5-bromo-N-(1-methylpiperidin-4-yl)pyridine-4-amine

To a stirred solution of 5-bromo-4-chloropyrimidine (300 mg, 1.562 mmol) in DMF, DIPEA (400 mg, 3.124 mmol) was added followed by 1-methylpiperidine-4-amine (0.193 ml, 1.562 mmol) and the reaction mixture was heated at 80° C. overnight. TLC showed absence SM, and then reaction mixture was concentrated. The crude product was purified by flash column chromatography over 230-400 mesh silica gel and using a mixture of 30% EtOAc/petroleum ether to afford the desired product 17, 180 mg (Yield: 40%) as yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 8.4 (s, 1H), 8.35 (s, 1H), 6.81 (d, 1H), 4.95 (m, 1H), 2.78 (d, 2H), 2.17 (s, 3H), 1.95 (t, 2H), 1.6-1.8 (m, 4H); MS-270 (M+1).

Example 14 5-(1-(4-methoxybenzyl)-3methyl-1H-pyrazolo[3,4-b]pyridine-5yl)-N-(1-methyl-piperidine-4yl)pyrazin-4-amine

To a stirred solution of 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (106 mg, 0.138 mmol) in toluene (2 ml), 5-bromo-N-(1-methylpiperid-4-yl)pyridine-4-amine (50 mg, 0.095 mmol), 2M sodium carbonate (0.2 ml), and EtOH (0.5 ml) were added. The reaction mixture was degassed with argon for 30 minutes. Tetrakis(triphenylphosphine)palladium (81 mg, 0.841 mmol) was added, degassed for another 30 minutes. Then RM was heated at 90° C. overnight. TLC was checked. Reaction mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate (50 ml). The extract was washed with water and concentrated. The crude product was purified by flash column chromatography over 230-400 mesh silica gel using 10% MeOH/DCM system to afford the desired product 18, 30 mg (Yield: 25%) as white solid. The product was confirmed by ¹HNMR and LCMS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 8.63 (s, 1H), 8.45 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.38 (d, 2H), 6.81 (d, 2H), 5.6 (s, 2H), 4.65 (bs, 1H), 4.17 (bs, 1H), 3.79 (s, 3H), 3.43 (s, 3H), 2.95 (bs, 1H), 2.59 (s, 3H), 22.22-2.45 (m, 5H), 2.13 (m, 2H); MS: 443, (M+1), LCMS˜95%.

Example 15 5-(3methyl-1H-pyrazolo[3,4-b]pyridine-5yl)-N-(1-methylpiperidine-4yl)pyrimidin-4-amine

To a stirred solution of 5-(1-(4-methoxybenzyl)-3methyl-1H-pyrazolo[3,4-b]pyridine-5yl)-N-(1-methylpiperidine-4yl)pyrazin-4-amine (150 mg) in DCM (2 ml) at 0° C., TFA (10 ml) was added and then heated at 60° C. overnight. TLC was checked. Reaction mixture was concentrated and basified with saturated sodium bicarbonate solution and again concentrated. The crude mass was purified by flash column chromatography over basic alumina using 1-3% MeOH/DCM as eluent to afford the desired product 19: 34 mg (Yield: 31.19%) as pale yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 10.64 (bs, 1H), 8.63 (s, 1H), 8.50 (s, 1H), 8.06 (s, 1H0, 8.03 (s, 1H), 4.67 (d, 1H), 4.075 (m, 1H), 2.89 (m, 2H), 2.61 (s, 3H), 2.29 (s, 3H), 2.24 (m, 2H), 2.04 (d, 2H); Ms: 324.31 (M+1), LCMS˜99.39%; HPLC ˜97.49%.

Example 16 Tetra-butyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridine-5-ylamino)piperidine-1-carboxylate

To a stirred solution of 3-Methyl-1H-pyrazole[3,4-b]pyridn-5-amine (500 mg, 3.3 mmol) in EtOH (50 ml), tert-butyl 3-oxopiperidine-1-carboxylate (74 mg, 1.1 mmol) was added and reaction mass was cooled to 0° C. Acetic acid (0.018 ml, 0.33 mmol) was added after which reaction mixture was stirred for 20 minutes. Sodium cyanoborohydride (42 mg, 0.66 mmol) was added and stirred for 12 h at RT. TLC showed absence of SM. The reaction mixture was quenched with brine solution and extracted with ethyl acetate. The extract was washed with water and concentrated. Organic layer was dried over anhydrous sodium sulphate, dried and concentrated. Product was purified by flash column chromatography over 230-400 mesh silica gel using 2-5% MeOH/DCM as eluent to afford the desired product 20, 500 mg (Yield: 45%) as white solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 8.13 (s, 1H), 7.19 (s, 1H), 3.95 (m, 1H), 3.25 (m, 1H), 3.43 (m, 1H), 3-3.25 (m, 2H), 2.52 (s, 3H), 2.15 (m, 1H), 1.78 (m, 1H), 1.6 (m, 2H), 1.49 (s, 9H); Ms-331 (M-56), LCMS˜97%.

Example 17 3-methyl-N-(piperidine-3yl)-1H-pyrazolo[3,4-b]pyridine-5-amine

To a stirred solution of tert-butyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridine-5-ylamino)piperidine-1-carboxylate (100 mg, 0.3 mmol) in DCM (5 ml) at 0° C., was added TFA (0.3 ml), and then reaction mixture was stirred for 2 h at RT. TLC showed absence of SM. The reaction mixture was concentrated and basified with saturated sodium bicarbonate solution, and extracted with ethyl acetate (3×25 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated. The product was purified by flash column chromatography over basic alumina using 1-5% MeOH/DCM as eluent to afford the desired product 21, 60 mg (Yield: 86%) as white solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 12.7 (d, 1H), 8.12 (s, 1H), 7.54 (s, 1H), 5.4 (m, 1H), 4.09 (m, 1H), 3.15 (m, 2H), 2.9 (dd, 1H), 2.6 (m, 1H), 2.45 (s, 3H), 1.95 (m, 1H), 1.72 (m, 1H), 1.05 (m, 1H), 1.4 (m, 1H), 1.35 (m, 1H); Ms: 231 (M+1), LCMS˜two peaks of same mass˜50%+38%.

Example 18 N-(1-(4-fluorobenzyl)piperidine-3-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridine-5-amine

To a suspension of 3-methyl-N-(piperidine-3yl)-1H-pyrazolo[3,4-b]pyridine-5-amine (60 mg, 0.259 mmol) in DCM (5 ml), TEA (0.04 ml, 0.311 mmol) was added and the reaction mixture was stirred for 20 min. Then to it 1-(bromomethyl)-4-fluorobenzene (58 mg, 0.311 mmol) was added and then the reaction mixture was stirred for 2 h at RT. TLC showed absence of SM. Reaction mixture was concentrated and the residue was purified by flash column chromatography over 230-400 mesh silica gel using 5-10% MeOH/DCM as eluent to afford the desired product 22, 70 mg (Yield: 28%) as pale yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 9.95 (bs, 1H), 8.05 (s, 1H), 2.27 (t, 2H), 6.99 (t, 2H), 4.11 (bs, 1H), 3.58-3.49 (m, 4H), 2.71 (m, 1H), 3.49 (s, 3H), 3.43 (m, 2H), 1.73 (m, 2H), 1.56 (bs, 2H); Ms: 339 (M+1), LCMS˜99.5%; HPLC˜98.27%.

Example 19 N-(1-(3,4-difluorobenzylpiperidin-3-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridid-5-amine

To a suspension of 3-methyl-N-(piperidine-3yl)-1H-pyrazolo[3,4-b]pyridine-5-amine (60 mg, 0.259 mmol) in DCM (5 ml) at rt, TEA (0.04 ml, 0.311 mmol) was added and the reaction mixture was stirred for 20 min. Then to this RM 1-(bromomethyl)-4-fluorobenzene (58 mg, 0.311 mmol) was added and was stirred for another 2 h at RT. TLC showed absence of SM. The reaction mixture was concentrated and was purified by flash column chromatography using 230-400 silica gel and 5-10% MeOH/DCM system to afford the desired product 26, 70 mg (Yield: 28%) as pale yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 9.83 (bs, 1H), 8.06 (d, 1H), 7.2-6.99 (m, 4H), 4.016 (m, 1H), 3.61 (m, 1H), 3.49-3.46 (m, 4H), 2.72 (m, 1H), 2.5-2.38 (m, 6H), 1.75 (m, 2H), 1.56 (m, 1H); Ms-357 (M+1), LCMS˜98.83%; HPLC˜98.5%.

Example 20 N-(1-(4-Chlorobenzyl)piperidin-3-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridid-5-amine

To a suspension of 3-methyl-1N-(piperidine-3yl)-1H-pyrazolo[3,4-b]pyridine-5-amine (70 mg, 0.262 mmol) in DCM (5 ml) at RT, TEA (0.052 ml, 0.386 mmol) was added and the reaction mixture was stirred for 20 min. Then to this RM 4-Chlorobenzylbromide (80 mg, 0.39 mmol) was added and was stirred for another 2 h at RT. TLC showed absence of SM. The reaction mixture was concentrated and was purified by flash column chromatography using 230-400 silica gel and 5-10% MeOH/DCM system to afford the desired product 27, 30 mg (Yield: 29%) as pale yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 10.25-10.1 (bs, 1H), 8.068 (s, 1H), 7.304-7.26 (m, 4H), 7.03 (s, 1H), 3.9-4.05 (bs, 1H), 3.58-3.49 (d, 3H), 2.7 (m, 1H), 2.5-2.3 (m, 6H), 1.7-1.6 (m, 4H); Ms: 357 (M+1), LCMS˜98.8%, HPLC˜98.48%.

Example 21 N-(1-(3,4-Chlorobenzyl)piperidin-3-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridid-5-amine

To a suspension of 3-methyl-1N-(piperidine-3yl)-1H-pyrazolo[3,4-b]pyridine-5-amine (75 mg, 0.324 mmol) in DCM (15 ml) at rt, TEA (0.056 ml, 0.422 mmol) was added and the reaction mixture was stirred for 20 min. Then to this RM 3,4-Dichlorobenzylbromide (101 mg, 0.422 mmol) was added and was stirred for another 2 h at RT. TLC showed absence of SM. The reaction mixture was concentrated and was purified by flash column chromatography over 230-400 silica gel using 5-10% MeOH/DCM as eluent to afford the desired product 28, 36 mg (Yield: 28%) as pale yellow solid. The product was confirmed by ¹HNMR and MS spectrum analysis. ¹H NMR (400 MHz, CDCl₃) δ: 10.1 (bs, 1H), 8.08 (d, 1H), 7.44 (bs, 1H), 7.38 (d, 1H), 7.17 (d, 1H), 7.04 (d, 1H), 4-3.9 (bs, 1H), 3.75 (d, 5H), 3.59 (m, 1H), 3.46 (s, 2H), 2.71 (m, 1H), 2.5 (s, 3H), 2.3 (m, 3H), 1.77 (m, 2H); Ms: 389, (M+1), LCMS˜97%; HPLC˜97%.

Example 22 2-Chloro-6-(2-(4-methylpiperazin-1-yl)methoxy)pyridine

To a suspension of 2-(4-methylpiperazine-1-yl)ethanol (50 mg, 0.347 mmol) in dioxane (3 ml) at 0° C., KO^(t)Bu (50 mg, 0.347 mmol) was added and the reaction mixture was stirred for 10 min. Ice bath was removed and the reaction mixture was allowed to attain room temperature. The mixture was again cooled to 0° C. and 2,6-Dichloropyrazine (200 mg, 1.04 mmol) was added. Reaction mixture was allowed to stir at RT for 24 h after which it was concentrated and was purified by flash column chromatography over 230-400 silica gel using 5-8% MeOH/DCM system to afford the desired product 31, 30 mg (Yield: 35%) as brown gummy liquid. The product was confirmed by ¹HNMR (not clean but characteristic peaks were present); MS: 256, (M+1), LCMS ˜90%.

Example 23 5-(6-(2-(3-methylimidazolidin-1-yl)ethoxy)pyarazin-2-yl)-1-(4-methoxy benzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a solution of 2-Chloro-6-(2-(4-methylpiperazin-1-yl)methoxy)pyridine (519 mg, 1.36 mmol) in acetonitrile, 1-(4-methoxybenzyl)-3methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (350 mg, 1.36 mmol) and 1M solution K₂CO₃ (2.46 mL, 2.46 mmol) was added. The reaction mixture was degassed for 20 min with nitrogen. Palladium acetate (10 mg, 0.041 mmol) and S-Phos (45 mg, 0.1 mmol) were added and reaction mixture was again degassed for 10 minutes and heated to 80° C. and maintained overnight. The reaction mixture was concentrated and purified by flash column by loading on a glass column packed with 230-400 mesh silica gel and eluted with 2-8% methanol/DCM. The product 32 was obtained as brown solid, 230 mg (Yield-35%) and confirmed by ¹HNMR and LCMS. ¹H NMR was not clean but characteristics peaks were present. MS: 473 (M+1), LCMS showed it as 82% pure.

Example 24 5-(6-(2-(4-methylpiperazin-1-yl)ethoxy)pyrazin-2-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 5-(6-(2-(3-methylimidazolidin-1-yl)ethoxy)pyarazin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (180 mg) in DCM (2 ml) TFA (8 ml) was added at 0° C. Ice-bath was removed and the mixture was heated to 50° C. and maintained for 16 h under stirring. Reaction mixture was cooled, concentrated and basified with saturated sodium bicarbonate solution, and again concentrated. The crude mixture was purified by flash column chromatography using basic alumina and 3% MeOH/DCM system to afford the desired product 33 as pale yellow solid, 60 mg (Yield: 47%) as pale yellow solid. The product was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 10.56 (bs, 1H), 9.2 (s, 1H), 8.65 (s, 1H), 8.60 (s, 1H), 8.22 (s, 1H), 4.63 (m, 2H), 2.919 (m, 2H), 2.67-2.54 (m, 11H), 2.33 (s, 3H); MS: 353, (M+1); LCMS˜98%, HPLC˜96%.

Example 25 N-(6-chloropyrazine-2-yl)-1-methylpiperidine-4-carboxamide

A solution of 1-methylpiperidine-4-carboxylic acid (500 mg, 3.1 mmol) in thionyl chloride (8 ml) was refluxed for 4 h. All volatiles are removed under nitrogen. To the crude acid chloride, pyridine (6 ml) was added followed by 2,6-Dichloropyrazine and the mixture was heated at 100° C. overnight. Reaction was completed as shown by TLC. Pyridine was removed under vacuum and the obtained crude product was purified by flash column over neutral alumina using 5% methanol/DCM as eluent. Compound 37 was obtained as dark brown solid, 130 mg (40%) and confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.06 (s, 1H), 9.30 (s, 1H), 8.46 (s, 1H), 2.77 (d, 2H), 2.58 (d, 2H), 2.15 (s, 3H), 1.86 (m, 4H), 1.8-1.74 (m, 2H); MS-254, (M+1), LCMS˜98%.

Example 26 N-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine-5-yl)pyrazin-2-oxamideyl)-1-methylpiperidine-4-carboxamide

To a solution of N-(6-chloropyrazine-2-yl)-1-methylpiperidine-4-carboxamide (100 mg, 0.39 momol) in DMF, 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (180 mg, 0.47 mmol) and 1M solution of Na₂CO₃ (0.4 mL) was added and the reaction mixture was degassed for 20 min with nitrogen. Pd₂dba₃ (36 mg, 0.039 mmol) and tri-t-butylphosphine (8 mg, 0.039 mmol) were added and degassed again for 10 minutes. The mixture was heated to 80° C. and maintained overnight. The reaction mixture was concentrated and purified by flash column using 230-400 mesh silica gel and 8% methanol/DCM system. The product 38 was obtained as brown solid, 60 mg (Yield-35%) and confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 9.50 (s, 1H), 9.14 (s, 1H), 8.84 (s, 1H), 8.53 (s, 1H), 7.96 (s, 1H), 7.35 (d, 2H), 6.83 (d, 2H), 5.61 (s, 2H), 3.76 (s, 3H), 3.0 (d, 2H), 2.62 (s, 3H), 2.38-2.3 (m, 3H), 2.02-1.95 (m, 7H); MS-471, (M+1), LCMS˜96%.

Example 27 1-methyl-N-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridine-5yl)pyrazin-2-yl)piperazine-4-carboxamide

A stirred solution of N-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine-5-yl)pyrazin-2-oxamideyl)-1-methylpiperidine-4-carboxamide (180 mg) in DCM (3 ml) was cooled to 0° C. Then TFA (10 ml) was added to it and heated to 60° C. and stirred overnight. TLC was checked. Reaction mixture was concentrated and basified with saturated sodium bicarbonate solution, and again concentrated. The crude product was purified by flash column chromatography using basic alumina eluting with 1-3% MeOH/DCM system to afford the desired product 39 as pale yellow solid, 60 mg (Yield: 30%). The product was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, DMSO) δ: 13.43 (s, 1H), 10.80 (s, 1H), 9.26 (d, 2H), 9.07 (s, 1H), 8.92 (s, 1H), 2.83 (d, 2H), 2.61-2.50 (m, 5H), 2.17 (s, 3H), 1.91-1.79 (m, 4H), 1.70-1.65 (m, 2H); MS: 351, (M+1), LCMS˜99%; HPLC˜98%.

Example 28 5-(6-chloropyrazin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of (1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine) 8 (105 mg, 0.276 mmol) and 2-bromo-6-chloropyrazine 40 (50 mg, 0.268 mmol, 1 equivalent) in DME (3 mL), ethanol (0.5 mL) and water (0.1 mL) was added sodium carbonate (2.0 M, 0.561 mmol, 2 equivalent) and degassed for 10 min followed by charging Pd(PPh₃)₄ (9.5 mg, 0.0082 mmol, 0.03 equivalent). The resulting reaction mixture was purged with N₂ and degassing was submitted for heating at 80° C. overnight under pressure in a sealed tube. After completion of the reaction the reaction was diluted with ethyl acetate and water. The organic layer was separated and aqueous phase was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulphate and evaporated. The crude product was flash chromatographed with 100-200 mesh silica gel eluting the pure compound at 40% ethyl acetate in hexane as off white coloured solid of compound 41 in 25 mg. The product 41 was confirmed by LCMS. MS: 365.9, (M+1).

Example 29 5-(6-chloropyrazin-2-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of compound 41 (20 mg) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) and heated at 50° C. overnight. After completion of the reaction the solvents were evaporated, diluted with cold water, pH was adjusted to 8-9 and the aqueous phase extracted with chloroform. The organic layer was washed with brine solution, dried over sodium sulphate and the solvents were evaporated to obtain the crude product. The crude material was passed through 100-200 mesh silica gel eluting to obtain the pure compound in 2% MeOH/CHCl₃ as pale yellow solid of compound 42 (5 mg). The product was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 9.16 (s, 1H), 9.01 (s, 1H), 8.72 (d, J=2.19 Hz, 1H), 8.57 (s, 1H), 2.66 (bs, 3H); LCMS. MS: 245.9, (M+1).

Example 30 1-(4-methoxybenzyl)-3-methyl-5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of compound 8 (230 mg, 0.627 mmol) and 2-bromopyrazine 43 (100 mg, 0.63 mmol) in DME (3 mL), ethanol (1 mL) and water (0.2 mL) was added sodium carbonate (135 mg, 1.26 mmol) and was degassed for 10 min followed by the charging Pd(PPh₃)₄ (7.5 mg, 0.03 equivalent). The resulting reaction mixture was purged with N₂ and degassing was submitted for heating at 80° C. overnight under pressure in a sealed tube. After completion of the reaction the reaction was diluted with ethyl acetate and water. The organic layer was separated and aqueous phase was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulphate and evaporated. The crude product was flash chromatographed with 100-200 mesh silica gel eluting the pure compound at 20% ethyl acetate in hexane as off white colored solid of compound 44 in 30 mg. The product 44 was confirmed by ¹H NMR (400 MHz, CDCl₃) δ: 9.18 (s, 1H), 9.17 (s, 1H), 9.10 (d, J=1.58 Hz, 2H), 8.67 (s, 1H), 8.64 (s, 1H), 7.35 (d, 2H), 6.84 (d, 1H), 5.62 (d, 2H), 3.76 (s, 3H), 2.62 (s, 3H).

Example 31 3-methyl-5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of compound 44 (30 mg) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) and heated at 50° C. overnight. After completion of the reaction the solvents were evaporated, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform. The organic layer was washed with brine solution, dried over sodium sulphate and the solvents were evaporated to obtain the crude product. The crude material was passed through 100-200 mesh silica gel eluting to obtain the pure compound in 1% MeOH/CHCl₃ as pale yellow solid of compound 45 (20 mg). The product was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, DMSO-d₆) δ: 13.42 (s, 1H), 9.39 (s, 1H), 9.25 (d, J=2.07, 1H), 8.95 (d, J=2.07, 1H), 8.74 (m, 1H), 8.62 (d, J=2.43, 1H), 2.57 (s, 3H); LCMS. MS: 211.9, (M+1).

Example 32 1-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)pyrrolidin-2-one

To a stirred solution of compound 8 (79 mg, 0.208 mmol) and compound 46 (50 mg, 0.208 mmol) in DME (3 mL), ethanol (1 mL) and water degassed and purged with nitrogen for 10 min was added sodium carbonate (45 mg, 0.420 mmol), again degassed for 10 min, then Pd(PPh₃)₄ (2.5 mg, 0.00207 mmol) was added and thoroughly degassed for another 10 min. The reaction mixture was heated at 80° C. overnight under sealed condition. After completion of the reaction the reaction mass was diluted with ethyl acetate and water and organic layer was separated and aqueous phase was again extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 40% ethyl acetate in hexane as off white coloured solid 47 (30 mg). MS m/z 415.0 (M+H)⁺.

Example 33 1-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)pyrrolidin-2-one

To a stirred solution of compound 47 (30 mg) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture heated at 50° C. overnight. After completion of the reaction the solvents was distilled off and diluted with cold water and pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 1-3% methanol in chloroform as pale yellow solid 10 mg of compound 48. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.49 (s, 1H), 9.27 (d, J=2.07, 1H), 9.10 (s, 1H), 8.95 (d, J=2.07, 1H), 4.16 (m, J=6.95, 7.31, 2H), 2.58 (s, 2H), 2.65 (m, 2H), 2.17 (m, 3H); MS m/z 294.9 (M+H)⁺.

Example 34 6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methyl pyrrolidin-3-yl)pyrazin-2-amine

To a stirred solution of 41 (158 mg, 0.432 mmol, 1.0 eq) and 49 (75 mg, 0.432 mmol, 1.0 eq) in 1,4 dioxane (5 mL) was added potassium tert-butoxide (97 mg, 0864 mmol, 2.0 eq) degas, the solution purged with nitrogen for 15 min, then was added Pd₂(dba)₃ (11 mg, 0.013 mmol, 0.03 eq) and BINAP (0.04 eq) and again degassed for 20 min. The reaction mixture was heated at 80° C. under sealed tube conditions overnight. After completion of the reaction the reaction mixture was diluted with water and extracted with dichloromethane twice. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound 50 at 90% ethyl acetate in hexane as pale yellow coloured compound 50 (50 mg).

Example 35 6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methylpyrrolidin-3-yl)pyrazin-2-amine

To a stirred solution of 50 (50 mg, 0.116 mmol, 1.0 eq) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture heated at 50° C. overnight. After completion of the reaction the reaction mixture the solvent was completely distilled off and diluted with cold water and pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) the pure compound at 1-3% methanol in chloroform as white solid 51 (20 mg). MS m/z 310.0 (M+H)⁺.

Example 36 2-chloro-6-(3,3-difluoropyrrolidin-1-yl)pyrazine

To a stirred solution of 52 (100 mg, 0.67 mmol, 1.0 eq) and 53 (80 mg, 0.73 mmol, 1.1 eq) in 1,4 dioxane (5 mL) was added DIPEA (216 mg, 1.34 eq, 2.0 eq) and the reaction mixture heated at 80° C. overnight. After completion of the reaction the reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and aqueous was again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 10% ethyl acetate in hexane to obtain 80 mg of compound 54. MS m/z 219.8 (M+H)⁺.

Example 37 5-(6-(3,3-difluoropyrrolidin-1-yl)pyrazin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 54 (80 mg, 0.364 mmol, 1.1 eq) and 8 (152 mg, 4.0 mml, 1.0 eq) in toluene (8 mL) and ethanol (2 ml) was added cesium carbonate (296 mg, 0.91 mmol, 2.5 eq) and the solution degassed and purged with nitrogen for 15 min, then Pd(PPh₃)₄ (4.2 mg, 0.0036 mmol, 0.01 eq) added and again the solution purged with nitrogen for another 15 min. The reaction mass was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction mixture was partitioned between ethyl acetate and water and the organic layer was separated and again extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off white coloured 54 compound as solid in 40 mg yield.

Example 38 5-(6-(3,3-difluoropyrrolidin-1-yl)pyrazin-2-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of compound 55 (40 mg, 0.0916 mmol, 1.0 eq) in dichloromethane (5 mL) was added trifluoroacetic acid (5 ml) and the reaction mixture heated at 50° C. overnight. After completion of the reaction the solvents were removed and diluted with cold water and pH was adjusted to 8 and extracted into the aqueous phase with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound 56 in 90% ethyl acetate in hexane as pale yellow solid compound 56 (5 mg). MS m/z 217.0 (M+H)⁺.

Example 39 2-(4-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)piperazin-1-yl)ethanol

To a stirred solution of 8 (86 mg, 0.226 mmol, 1.1 eq) and 57 (50 mg, 0.206 mmol, 1.0 eq) in toluene (2 mL) and ethanol (0.1 ml) was added cesium carbonate (127 mg, 0.0389 mmol, 2.0 eq) with degassing and purging with nitrogen for 15 min, then was added Pd(PPh₃)₄ (2.3 mg, 0.00206 mmol, 0.01 eq). The reaction mixture was purged again with N₂ for another 15 min and was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off-white coloured solid 58 in 25 mg quantity.

Example 40 2-(4-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)piperazin-1-yl)ethanol

To a stirred solution of 58 (25 mg, 0.054 mmol, 10 eq) in dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) and the resulting reaction mixture heated at 50° C. overnight. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 35-40% ethyl acetate in hexane as pale yellow solid compound 59 in 5 mg quantity. MS m/z 340.1 (M+H)⁺.

Example 41 (6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)(4-methylpiperazin-1-yl)methanone

To a stirred solution of compound 8 and 60 in toluene and ethanol was added cesium carbonate, the solution degassed and purged with nitrogen for 15 min, then Pd(PPh3)4 added and again purged with nitrogen for another 15 min. The reaction mass was heated at 100° C. in a sealed tube overnight. After completion of the reaction, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and again extract with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 13% ethyl acetate in hexane as off-white colored solid 61 (100 mg).

Example 42 (6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)(4-methylpiperazin-1-yl)methanone

To a stirred solution of 61 in dichloromethane were added trifluoroacetic acid and the reaction mixture heated at 50° C. overnight. After completion of the reaction the solvents were distilled off, diluted with cold water, pH was adjusted to 8-9 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and solvents evaporated to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 26% ethyl acetate in hexane as pale yellow solid of compound 62 in 25 mg. The spectral data shows that the compound is de-methylated compound 62. MS m/z 323.4 (M+H)⁺.

Example 43 1-(4-methoxybenzyl)-3-methyl-5-(6-(4-methylpiperazin-1-yl)pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 8 and 63 in 1,4 dioxane and water was added Cs₂CO₃, the solution degassed, purged with nitrogen for 15 min, Pd(PPh₃)₄ was added and the reaction mass was again purged with nitrogen for another 15 min. The reaction mass was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction mass was partitioned between ethyl acetate and water and the organic layer was separated and again extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 70-80% ethyl acetate in hexane as off white coloured solid compound 64 (25 mg).

Example 44 3-methyl-5-(6-(4-methylpiperazin-1-yl)pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 64 in dichloromethane was added trifluoroacetic acid and the reaction mixture heated to 50° C. for 12 hours. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 2% MeOH/CHCl₃ as pale yellow solid (10 mg) compound 65. MS m/z 310.4 (M+H)⁺.

Example 45 N¹-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)-N²,N²-dimethylethane-1,2-diamine

To a stirred solution of 8 and 66 in 1,4-dioxane and CH₂Cl₂ was added Cs₂CO₃, the solution degassed, purged under N₂ for 15 min, Pd(dppf)Cl₂ was added followed by purging again with N₂ for another 15 min. The reaction mass was heated at 100° C. in a sealed tube overnight. After completion of the reaction it was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and the solvent distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 1-2% of methanol in chloroform as off-white colored solid compound 67 (40 mg).

Example 46 N¹,N¹-dimethyl-N2-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)ethane-1,2-diamine

To a stirred solution of 67 in chloroform was added trifluoroacetic acid and the reaction mixture heated at 50° C. overnight. After completion of the reaction the solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting the pure compound 68 at 2% MeOH/CHCl₃ as pale yellow solid (10 mg). MS m/z 298.0 (M+H)⁺.

Example 47 6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine

To a stirred solution of 8 and 69 in toluene and ethanol was added Cs₂CO₃, the solution degassed, purged with N₂ for 15 min then Pd (PPh₃)₄ added and the solution again purged with nitrogen for another 15 min. The reaction mass was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off-white colored solid 70 (30 mg).

Example 48 N-methyl-6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine

To a stirred solution of 70 in chloroform was added trifluoroacetic acid and the mixture heated at 50° C. overnight. After completion of the reaction the solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting the pure compound 71 at with 40% EtOAc and hexane to yield pale yellow solid compound 71 (10 mg). MS m/z 339.13 (M+H)⁺.

Example 49 1-(4-methoxybenzyl)-3-methyl-5-(3-(pyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 8 and 72 in dimethoxyethane was added Cs₂CO₃. The reaction mixture was degassed, purged with N₂ for 15 min Pd (PPh₃)₄ was added, the mixture purged again for another 15 min. The resulting reaction mixture was heated at 100° C. in a sealed tube overnight. After completion of the reaction, the mixture was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off-white coloured solid 73 (30 mg).

Example 50 3-methyl-5-(3-(pyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 73 in chloroform was added trifluoroacetic acid and heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting with 70% EtOAc and hexane to yield pale yellow solid compound 74 (15 mg). MS m/z 280.93 (M+H)⁺.

Example 51 (6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)(piperidin-1-yl)methanone

To a stirred solution of 8 and 75 in dimethoxyethane was added Cs₂CO₃. The reaction mixture was degassed, purged with N₂ for 15 min Pd (PPh₃)₄ was added, purged again for another 15 min. The resulting reaction mixture was heated at 100° C. in a sealed tube for 12 hours. After completion of the reaction, the mixture was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off-white coloured solid 76 (60 mg).

Example 52 (6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)(piperidin-1-yl)metha-none

To a stirred solution of 76 in chloroform was added trifluoroacetic acid and the solution heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting with 60% EtOAc and hexane to yield pale yellow solid compound 77 (15 mg). MS m/z 323.00 (M+H)⁺.

Example 53 2-(4-((6-chloropyrazin-2-yl)amino)piperidin-1-yl)ethanol

To a stirred solution of 52 and 2-(4-aminopiperidin-1-yl)ethanol in DMF was added DIPEA and the reaction mixture was heated at 100° C. overnight under sealed conditions. After completion of the reaction the reaction was partitioned between water and DCM, the aqueous phase was separated and extracted with DCM again. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting with 10% methanol in chloroform to yield 30 mg of compound 79.

Example 54 2-(4-((6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)amino)piperidin-1-yl)ethanol

To a stirred solution of 41 and 79 in 1,4-dioxane was added rac-BINAP and tert-BuOK. The reaction mixture was degassed, purged with N₂ for 15 min Pd (dba)₃ was added, the mixture was purged again for another 15 min. The resulting reaction mixture was heated to 80° C. in sealed tube for 12 hours. After completion of the reaction, the solvents were removed and extracted with dichloromethane. The organic layers were dried over sodium sulphate and distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting with 1-2% MeOH and CHCl₃ as off-white coloured solid 80 (45 mg).

Example 55 2-(4-((6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)amino)piperidin-1-yl)ethanol

To a stirred solution of 80 in chloroform was added trifluoroacetic acid and the mixture heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CH₂Cl₂ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through silica gel eluting with 1-2% MeOH and CHCl₃ to yield pale yellow solid compound 81 (45 mg). MS m/z 352.9 (M+H)⁺.

Example 56 4-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)piperazin-2-one

To a stirred solution of 8 and 132 in dimethoxyethane was added Na₂CO₃. The reaction mixture was degassed, purged with N₂ for 15 min Pd (PPh₃)₄ was added, the mixture purged again for another 15 min. The resulting reaction mixture was heated at 100° C. in a sealed tube for 10 hours. After completion of the reaction, the mixture was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% ethyl acetate in hexane as off-white coloured solid 82 (15 mg).

Example 57 4-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)piperazin-2-one

To a stirred solution of 82 in chloroform was added trifluoroacetic acid and the mixture heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CH₂Cl₂ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting with 3% MeOH and CHCl₃ to yield pale yellow solid compound 83 (15 mg).

Example 58 6-chloro-N-(1-isopropylpiperidin-4-yl)pyrazin-2-amine

To a stirred solution of 52 and 84 in DMF was added DIPEA and the reaction mixture was heated at 100° C. overnight under sealed conditions. After completion of the reaction the reaction was partitioned between water and DCM, the aqueous phase was separated and extracted with DCM again. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 8% methanol in chloroform to yield 80 mg of compound 85.

Example 59 N-(1-isopropylpiperidin-4-yl)-6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-amine

To a stirred solution of 8 and 86 in DMF was added Cs₂CO₃. The reaction mixture was degassed, purged with N₂ for 15 min and Pd(dppf)Cl₂ was added. The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and again extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvent distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 6% MeOH/CHCl₃ (30 mg) to give compound 86.

Example 60 N-(1-isopropylpiperidin-4-yl)-6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-amine

To a stirred solution of 86 in CHCl₃ was added trifluoroacetic acid. The mixture was heated at 50° C. overnight. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through neutral alumina eluting the pure compound at 3% MeOH/CHCl₃ as pale yellow solid 87 (10 mg). MS m/z 352.1 (M+H)⁺.

Example 61 5-(6-(indolin-1-yl)pyrazin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 8 and 88 in 1,2-dimethoxyethane was added water, Cs₂CO₃ followed by degasssing, purging with N₂ for 15 min and addition of Pd(PPh₃)₄. The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction mass was partitioned between ethyl acetate and water and the organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 50% ethyl acetate in hexane as off-white coloured solid compound 89 (40 mg).

Example 62 5-(6-(indolin-1-yl)pyrazin-2-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 89 in chloroform was added trifluoroacetic acid and the mixture heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CH₂Cl₂ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through aluminium oxide (neutral) eluting with 30% EtOAc and hexane to yield pale yellow solid compound 90 (15 mg). MS m/z 329.0 (M+H)⁺.

Example 63 1-(3-((6-chloropyrazin-2-yl)amino)phenyl)pyrrolidin-2-one

To a stirred solution of 52 and 91 in toluene was added Pd(OAc)₂, Cs₂CO₃ and the resulting reaction mixture was degassed, purged with N₂, racemic BINAP was added and the reaction mixture was heated at 100° C. overnight under sealed conditions. After completion of the reaction the solvents were removed and the product was passed through 100-200 mesh silica gel eluting the pure compound at 8% methanol in DCM to obtain the intermediate 92 (10 mg).

Example 64 1-(3-((6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)amino)phenyl)pyrrolidin-2-one

To a stirred solution of 8 and 92 in 1,2-dimethoxyethane was added Cs₂CO₃ followed by degassing, purging with N₂ for 15 min and addition of Pd(PPh₃)₄. The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion of the reaction the reaction mass was partitioned between ethyl acetate and water and the organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 50% ethyl acetate in hexane as off-white coloured solid compound 93 (40 mg).

Example 65 1-(3-((6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)amino)phenyl)pyrrolidin-2-one

To a stirred solution of 93 in chloroform was added trifluoroacetic acid and the mixture heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CHCl₃ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through neutral alumina eluting the pure compound at 4% MeOH/CHCl₃ as pale yellow solid 94 (20 mg). MS m/z 386.1 (M+H)⁺.

Example 66 5-(3-chlorophenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (100 mg, 0.301 mmol, 1 eq) and 2-(3-chlorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (95) (71 mg, 0.301 mmol, 1 eq) in 1,2-dimethoxyethane (10 mL) was added Cs₂CO₃ (244 mg, 0.752 mmol, 25 eq) 2M aqueous solution followed by degassing, purging with N₂ for 15 min and addition of Pd(PPh₃)₄ (13 mg, 0.012 mmol, 0.04 eq). The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion of the reaction the mixture was partitioned between ethyl acetate and water and the organic layer was separated and again extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and the solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 50% ethyl acetate in hexane as off-white coloured solid compound 96 (60 mg).

Example 67 N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)-1-methylpiperidin-4-amine

To a stirred solution of 5-(3-chlorophenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (96) (50 mg, 0.137 mmol) in tert-BuOH (4 mL), 1,4 dioxane (6 mL) was added 1-methylpiperidin-4-amine (16) (14 mg, 0.23 mmol, 0.9 eq) and tert-BuOK (38 mg, 0.344 mmol, 2.5 eq). The resulting reaction mixture was degassed, purged with N₂ for 10 min and Pd₂(dba)₃ (5 mg, 0.00549 mmol, 0.04 eq) and tertButyl X-phos (0.08 eq) was added, the mixture was degassed and purged with nitrogen for 15 min. The reaction mixture was stirred overnight at 80° C. under sealed condition for 16 h. After completion of the reaction the reaction mass was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and solvent completely distilled off to get the crude. The crude was passed through 100-200 mesh silica gel. Eluting the pure compound at 5% MeOH/CHCl₃ gave 25 mg of N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)-1-methylpiperidin-4-amine 97.

Example 68 1-methyl-N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)piperidin-4-amine

To a stirred solution of N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)-1-methylpiperidin-4-amine (97) (25 mg, 0.056 mmol) in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the mixture was heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CHCl₃ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through neutral alumina eluting the pure compound at 2% MeOH/CHCl₃ as pale yellow solid 1-methyl-N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)piperidin-4-amine 98 (8 mg).

Example 69 5-(6-chloropyridin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (139 mg, 0.418 mmol) in 1,2-dimethoxyethane (10 mL) was added 2-chloro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (99) (100 mg, 0.418 mmol) and Cs₂CO₃ (32 mg, 0.104 mmol). The resulting reaction mixture was degassed, purged with N₂ for 15 min, Pd(PPh₃)₄ (19 mg, 0.0167 mmol) was added and the mixture purged again under nitrogen for another 15 min. The reaction mass was heated at 100° C. in a sealed tube for 12 h. After completion of the reaction the reaction mass was partitioned between ethyl acetate and water and the organic layer was separated and extracted with ethyl acetate. The combined organic layer was dried over Na₂SO₄ and the solvent completely distilled off to get the crude product. The crude material was passed through 100-200 mesh silica gel eluting the pure compound at 50% ethyl acetate in hexane to yield off white colored solid compound 5-(6-chloropyridin-2-yl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine 100 (60 mg).

Example 70 6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methyl-piperidin-4-yl)pyridin-2-amine

To a stirred solution of 5-(3-chlorophenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (96) (60 mg, 0.164 mmol, 1 eq) and 1-methylpiperidin-4-amine (16) (81 mg, 0.164 mmol, 1.0 eq) in 1,4 dioxane (10 mL) was added tert-BuOK (46 mg, 0.411 mmol, 2.5 eq), the mixture was then degassed and purged with nitrogen for 10 min. Pd₂(dba)₃ (5 mg, 0.00549 mmol, 0.04 eq) and racemic BINAP (0.03 eq) was added and the reaction mixture again degassed and purged again with nitrogen for 15 min. The reaction mass was stirred overnight at 80° C. under sealed condition. After completion of the reaction the reaction mass was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and the solvents were removed to get the crude material which was passed through 100-200 mesh silica gel eluting the pure 6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyridin-2-amine 101 at 2% MeOH/CHCl3 to yield 30 mg.

Example 71 6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyridin-2-amine

To a stirred solution of 6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyridin-2-amine (101) (25 mg, 0.0678 mmol) in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the mixture heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CHCl₃ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through neutral alumina eluting the pure compound at 2% MeOH/CHCl₃ as pale yellow solid 6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N-(1-methyl-piperidin-4-yl)pyridin-2-amine 102 obtained in 8 mg yield.

Example 72 4-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)morpholine

To a stirred solution of 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (8) (190 mg, 0.50 mmol, 1 eq) and 4-(6-chloropyrazin-2-yl)morpholine (103) (100 mg, 0.50 mmol, 1 eq) in 1,2-dimethoxyethane (5 mL) was added CsCO₃ (400 mg, 2.5 eq) in 2M H₂O and the resulting reaction mixture was degassed and purged with nitrogen for 10 min. Pd(PPh)₃ (0.023 mg, 0.04 mmol) was charged and the reaction mixture degassed and purged again with nitrogen for another 15 min. The reaction mass was stirred overnight at 100° C. under sealed condition 12 hrs. After completion of the reaction the reaction mass was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and the solvents were removed to get the crude material which was passed through 100-200 mesh silica gel eluting the pure 4-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)morpholine 104 at 50% EtOAc in hexane, providing off-white solid in yield of 60 mg.

Example 73 4-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)morpholine

To a stirred solution of 4-(6-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)morpholine (104) (60 mg, 0.144 mmol) in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. overnight. The solvents were removed and diluted with cold water and pH was adjusted to 8, the aqueous phase extracted with CHCl₃ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the compound at 50% ethyl acetate in hexane as pale yellow solid 4-(6-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2-yl)morpholine 105 in 30 mg quantity. MS m/z 296.9 (M+H)⁺.

Example 74 1-(4-methoxybenzyl)-3-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (8) (100 mg, 0.302 mmol, 1 eq) and 4-bromo-1-methyl-1H-pyrazole (106) (62 mg, 0.302 mmol, 1 eq) in 1,2-dimethoxyethane (8 mL) was added Cs₂CO₃ (325.18 mg, 0.604 mmol, 2 eq), the reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (10 mg, 0.009, 0.03 eq) was added and the reaction mixture again purged under N₂ for another 15 min. The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion, the reaction mixture was partitioned between ethyl acetate/water; the organic layer was separated and extracted again into ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 25% EtOAc/Hexane as off-white coloured solid 1-(4-methoxybenzyl)-3-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine 107 (60 mg).

Example 75 3-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 1-(4-methoxybenzyl)-3-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine (107) (60 mg, 0.179 mmol) dissolved in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 80% ethyl acetate in hexane as pale yellow solid 3-methyl-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine 108 (30 mg).

Example 76 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N,N-dimethyl benzenesulfonamide

To a stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (100 mg, 0.302 mmol, 1 eq) and (3-(N,N-dimethylsulfamoyl)phenyl)boronic acid (109) (54 mg, 0.302 mmol, 1 eq) in 1,2-dimethoxyethane (6 mL) was added Cs₂CO₃ (189 mg, 0.604 mmol, 2 eq), the reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (10 mg, 0.008, 0.03 eq) was added and again purged under N₂ for another 15 min. The reaction mixture was heated to 100° C. in sealed tube for 16 hrs. After completion, the reaction mixture was partitioned between ethyl acetate/water; the organic layer was separated and extracted again into ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 40% EtOAc/Hexane as off-white colored solid 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N,N-dimethyl benzenesulfonamide 110 (50 mg).

Example 77 N,N-dimethyl-3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzenesulfonamide

To a solution of 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)-N,N-dimethylbenzene-sulfonamide (110) (50 mg, 0.114 mmol) dissolved in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 70% ethyl acetate in hexane as pale yellow solid N,N-dimethyl-3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzenesulfonamide 111 (30 mg). MS m/z 316.9 (M+H)⁺.

Example 78 N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetamide

To a stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (160 mg, 0.505 mmol, 1 eq) and (3-acetamidophenyl)boronic acid (112) (100 mg, 0.505 mmol, 1 eq) in 1,2-dimethoxyethane (6 mL) was added Cs₂CO₃ (246 mg, 0.750 mmol, 1.5 eq) and Pd₂(OAc), the reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (10 mg, 0.008, 0.03 eq) was added and the mixture was again purged under N₂ for another 15 min. The reaction mixture was heated to 100° C. in a sealed tube for 16 hrs. After completion, the reaction mixture was partitioned between ethyl acetate/water; the organic layer was separated and extracted again into ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 40% EtOAc/Hexane as off-white coloured solid N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetamide 113 (50 mg).

Example 79 N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetamide

To a solution of N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetamide (113) (50 mg, 0.129 mmol) dissolved in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 40% ethyl acetate in hexane as pale yellow solid N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetamide 114 (30 mg). MS m/z 266.9 (M+H)⁺.

Example 80 1-(4-methoxybenzyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 1-(4-methoxybenzyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (115) (100 mg, 0.302 mmol, 1 eq) and 4-bromo-1-methyl-1H-pyrazole (106) (54 mg, 0.302 mmol, 1 eq) in 1,2-dimethoxyethane (6 mL) was added Cs₂CO₃ (189 mg, 0.604 mmol, 2 eq) and Pd₂(OAc) (0.03 eq), the reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (17 mg, 0.0151, 0.03 eq) was added and the reaction mixture again purged under N₂ for another 15 min. The reaction mixture was heated to 100° C. in a sealed tube for 16 hrs. After completion, the reaction mixture was partitioned between ethyl acetate/water; the organic layer was separated and extracted again into ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 25% EtOAc/Hexane as off-white coloured solid 1-(4-methoxybenzyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine 116 (40 mg).

Example 81 5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 1-(4-methoxybenzyl)-5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine (116) (40 mg, 0.125 mmol) dissolved in chloroform (10 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 40% ethyl acetate in hexane as pale yellow solid of 5-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridine 117 obtained in 10 mg quantity. MS m/z 199.9 (M+H)⁺.

Example 82 Ethyl 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

To a stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (171 mg, 0.515 mmol, 1 eq) and (3-(ethoxycarbonyl)phenyl)boronic acid (100 mg, 0.515 mmol, 1 eq) in 1,2-dimethoxyethane (6 mL) was added Cs₂CO₃ (335 mg, 1.03 mmol, 2.0 eq). The reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (0.0238 mg, 0.0206, 0.04 eq) was added and the reaction mixture again purged under N₂ for another 15 min. The reaction mixture was heated at 100° C. in a sealed tube for 16 hrs. After completion, the reaction was partitioned between ethyl acetate/water; the organic layer was separated and extracted again into ethyl acetate. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 25% EtOAc/Hexane as off-white coloured solid ethyl 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate 118 (70 mg).

Example 83 Ethyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

To a solution of 3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (118) (70 mg, 0.74 mmol) dissolved in chloroform (5 mL) was added trifluoroacetic acid (8 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 40% ethyl acetate in hexane as pale yellow solid of ethyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate 119 obtained in 10 mg quantity. MS m/z 281.9 (M+H)⁺.

Example 84 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoic acid

To a stirred solution of ethyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (119) (10 mg, 0.142, 1 eq) in ethanol (5 mL) was added 4N sodium hydroxide solution (4 mL) and the reaction mixture was stirred at room temperature for 4 hrs. After completion of the reaction the solvents were distilled off and diluted with water and the pH adjusted to 4 and extracted into chloroform. The organic layer was dried over sodium sulphate and the solvents were removed to get the compound 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoic acid 120 and this was further titrated with hexane and diethyl ether to get 15 mg of 120.

Example 85 1-(4-methoxybenzyl)-3-methyl-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 1-(4-methoxybenzyl)-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (8) (100 mg, 0.263 mmol, 1 eq) and 1-(4-bromobenzyl)-4-methylpiperazine (121) (77 mg, 0.29 mmol, 1 eq) in 1,2-dimethoxyethane (10 mL) was added Cs₂CO₃ (257 mg, 0.791 mmol, 3 eq), the reaction mixture was degassed and purged under N₂ for 15 min and Pd(PPh3)₄ (12 mg, 0.00105, 0.04 eq) was added and again the reaction mixture was purged under N₂ for another 15 min. The reaction mixture was heated at 100° C. in a sealed tube overnight. After completion, the reaction mixture was partitioned between CHCl₃/H₂O; the organic layer was separated and extracted again into CHCl₃. The combined organic layer was dried over sodium sulphate and the solvents were removed to get the crude product. The crude material was flashed through 100-200 mesh silica gel eluting the pure compound in 6% MeOH/CHCl₃ as off-white coloured solid 1-(4-methoxybenzyl)-3-methyl-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridine 122 (50 mg). MS m/z 442.1 (M+H)⁺.

Example 86 3-methyl-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 1-(4-methoxybenzyl)-3-methyl-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridine (122) (30 mg, 0.136 mmol) dissolved in chloroform (25 mL) was added trifluoroacetic acid (6 mL) and the reaction mixture was heated at 50° C. for 12 h. After completion of the reaction the solvents were removed and diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform two times. The organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed. The crude was passed through 100-200 mesh silica gel eluting the compound at 7% MeOH/CHCl₃ as pale yellow solid of 3-methyl-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridine 123 obtained in 10 mg quantity. MS m/z 322.1 (M+H)⁺.

General Procedures for the Schemes 31-46

The key building block 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine 124 (1 eq), 2-substituted heteroaryl halogen containing scaffolds (1.0 eq) in either DMF, DME, toluene, acetonitrile, tert-BuOH, THF and or 1,4-dioxane was taken in a sealed tube. The resulting reaction mixture was degassed, purged with argon or N₂ gas a few times and was charged with NaCO₃, Cs₂CO₃, K₂CO₃, potassium acetate or NaHCO₃ (between 1.5 to 2 equivalents) followed by the addition of palladium catalysts (0.01 to 0.05 eq). After the addition of catalysts the contents of the reaction were purged and degassed again and the reaction mixture was heated at between 80 to 100° C. for 8 to 16 hrs. After completion of the reaction monitored from TL, the contents were cooled to room temperature and diluted with CH₂Cl₂, CHCl₃ or EtOAc. The organic layers were passed through a Celite pad then the solvent was completely distilled off to get the crude product. The crude was subjected to flash column chromatography purification to get the title compounds.

Example 87 5-(6-(4-methylpiperazin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.2 mmol, 1 eq) and 2-chloro-6-(4-methylpiperazin-1-yl)pyrazine (63) (44 mg, 0.2 mmol, 1 eq) in DMF (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.4 mmol, 2 eq) followed by addition of Pd(PPh₃)₄ (0.01 mg, 0.04 mmol) and degassing and purging under argon for an additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 2% MeOH/CHCl₃ to obtain pale yellow solid compound 5-(6-(4-methylpiperazin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine 125 in 20 mg quantity. The compound 125 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 8.96 (d, J=1.5 Hz 1H), 8.55 (d, J=1.70 Hz, 1H), 8.30 (s, 1H), 8.08 (s, 1H), 7.35 (m, J=3.04, 1H), 6.60 (m, J=1.95 1H), 3.75 (m, J=5.00, 4H), 2.58 (m, J=5.12, 4H), 2.38 (s, 3H); MS m/z 294.9 (M+H)⁺.

Example 88 2-(4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)piperazin-1-yl)ethanol

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.2 mmol, 1 eq) and 2-(4-(6-chloropyrazin-2-yl)piperazin-1-yl)ethanol (57) (49.5 mg, 0.2 mmol, 1 eq) in DME (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.4 mmol, 2 eq) followed by addition of Pd(PPh₃)₄ (1.0 mg, 0.04 mmol) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 3% MeOH/CHCl₃ to obtain pale yellow solid compound 2-(4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)piperazin-1-yl)ethanol 126 in 15 mg quantity. The compound 126 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 8.96 (s, 1H), 8.90 (s, 1H), 8.54 (s, 1H), 8.40 (s, 1H), 8.09 (s, 1H), 7.36 (m, J=289.99, 1H), 6.61 (m, J=301.8, 1H), 3.75 (m, J=5.0, 6H), 2.69 (m, J=4.84, 6H); MS m/z 324.9 (M+H)⁺.

Example 89 N1-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)-N2,N2-dimethylethane-1,2-diamine

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (70 mg, 0.286 mmol, 1 eq) and N1-(6-chloropyrazin-2-yl)-N2,N2-dimethylethane-1,2-diamine (66) (457 mg, 0.286 mmol, 1 eq) in 1,4-dioxane (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (150 mg, 0.614 mmol, 1.5 eq) followed by addition of Pd(dpp)Cl₂ (6.2 mg, 0.0086 mmol, 0.03 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through oCelite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound N1-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)-N2,N2-dimethylethane-1,2-diamine 127 in 10 mg quantity. The compound 127 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.78 (s, 1H), 8.90 (d, J=1.82, 1H), 8.57 (s, 1H), 8.30 (s, 1H), 7.89 (s, 1H), 7.51 (m, J=151.58, 1H), 7.01 (m, J=201.21, 1H), 6.53 (m, J=191.45, 1H), 3.48 (m, J=5.85, 2H), 2.22 (s, 6-H); MS m/z 283 (M+H)⁺.

Example 90 5-(3-(pyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.2048 mmol, 1 eq) and 2-chloro-3-(pyrrolidin-1-yl)pyrazine (72) (33 mg, 0.184 mmol, 1 eq) in DME (8 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.409 mmol, 2 eq) followed by addition of Pd(dpp)Cl₂ (6 mg, 0.00819 mmol, 0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound 5-(3-(pyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine 128 in 10 mg quantity. The compound 128 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 9.50 (s, 1H), 8.58 (d, J=1.95, 1H), 8.15 (d, J=1.58, 1H), 8.04 (m, J=2.56, 2H), 7.38 (m, J=3.45, 1H), 6.56 (m, J=1.58, 1H), 3.18 (m, J=6.70, 1H), 1.81 (m, J=2.92, 4H; MS m/z 265.9 (M+H)⁺.

Example 91 N-(1-methylazetidin-3-yl)-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-amine

To the starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and 2-chloro-6-((1-methylazetidin-3-yl)oxy)pyrazine (129) (40.9 mg, 0.410 mmol, 1 eq) in 1,4-dioxane was added Pd(dpp)Cl₂ (1.184 mg, 0.1024 mmol, 0.005 eq) and Cs₂CO₃ (2 eq) followed by degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound N-(1-methylazetidin-3-yl)-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-amine 130 in 16 mg quantity.

2-chloro-6-((1-methylazetidin-3-yl)oxy)pyrazine (129) was prepared by reacting 1-methylazetidin-3-ol (50 mg, 0.336 mmol, 1 eq) and 2,6-dichloropyrazine (32) (29.2 mg, 0.336 mmol, 1 eq) in DMF (5 mL) and sodium hydride (16.11 mg, 0.671 mmol, 2 eq) under refluxing temperature for 8 hrs.

Example 92 1-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)pyrrolidin-2-one

To the starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and 1-(6-bromopyrazin-2-yl)pyrrolidin-2-one (46) (49 mg, 0.205 mmol, 1 eq) in DME (5 mL), was added Pd(PPh₃)₄ (7.1 mg, 0.00614 mmol, 0.02 eq) Cs₂CO₃ (133 mg, 0.410 mmol, 2 eq) and degassing and purging under argon for additional 10 min was performed. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound 1-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)pyrrolidin-2-one 131 in 9 mg quantity. The compound 131 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.90 (s, 1H), 9.46 (s, 1H), 9.02 (d, J=10.48, 2H), 8.71 (s, 1H), 7.56 (s, 1H), 6.58 (s, 1H)], 4.13 (m, J=7.07, 2H), 2.65 (m, J=8.04, 2H), 2.14 (m, J=192.19, 2H); MS m/z 279.9 (M+H)⁺.

Example 93 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)piperazin-2-one

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and 4-(6-chloropyrazin-2-yl)piperazin-2-one (132) (33 mg, 0.184 mmol, 0.9 eq) in DME (8 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.409 mmol, 2 eq) followed by addition of Pd(dpp)Cl₂ (6 mg, 0.00819 mmol, 0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)piperazin-2-one 133 in 9 mg quantity. The compound 133 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.84 (s, 1H), 8.95 (d, J=2.07, 1H), 8.64 (d, J=1.95, 1H), 8.57 (s, 1H), 8.23 (s, 1H), 8.18 (s, 1H, 7.53 (m, J=2.92, 1H), 6.56 (m, J=1.70, 1H), 4.18 (s, 2H), 3.89 (m, J=5.36 2H); MS m/z 294.9 (M+H)⁺.

Example 94 5-(6-(indolin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine

The starting material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and 1-(6-chloropyrazin-2-yl)indoline (88) 423 mg, 0.185 mmol, 0.9 eq) in DME (7 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.410 mmol, 2 eq) followed by addition of Pd(dpp)Cl₂ (6 mg, 0.00819 mmol, 0.04 eq) and degassing and purging under argon for additional 10 minutes was performed. The reaction mixture was heated at 90° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound 5-(6-(indolin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine 134 in 12 mg quantity. The compound 134 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 9.06 (s, 2H), 8.59 (s, 1H), 8.60 (s, 1H), 8.55 (s, 1H), 8.44 (m, 1H), 8.16 (s, 1H), 7.41 (m, 1H), 6.99 (m, 1H), 4.21 (m, J=8.53, 2H), 3.33 (m, J=341.82, 2H); MS m/z 313.9 (M+H)⁺.

Example 95 5-(6-(3,3-difluoropyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 1 eq) and 2-chloro-6-(3,3-difluoropyrrolidin-1-yl)pyrazine (54) (1 eq) in 1,4-dioxane (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (2 eq) followed by addition of Pd(dpp)Cl₂ (0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound 5-(6-(3,3-difluoropyrrolidin-1-yl)pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine 135 in 6 mg quantity.

Example 96 1-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)piperidin-4-ol

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and 1-(6-chloropyrazin-2-yl)piperidin-4-ol (136) (44 mg, 0.205 mmol, 1 eq) in 1,2-dimethoxyethane (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.410 mmol, 2 eq) followed by addition of Pd(dpp)Cl₂ (0.006 mg, 0.00819 mmol, 0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 90° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound (6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)(piperidin-1-yl)methanone 137 in 12 mg quantity. The compound 137 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.81 (s, 1H), 8.91 (d, J=2.07, 1H), 8.60 (d, J=2.07, 1H), 8.45 (s, 1H), 8.26 (s, 1H), 7.52 (m, J=2.56, 1H), 6.55 (m, J=1.82, 1H), 4.74 (m, J=4.26 1H), 4.14 (m, J=13.65, 2H), 3.75 (m, J=4.14, 1H), 3.34 (m, J=163.12, 1H), 3.26 (m, J=11.21, 2H), 1.84 (m, J=9.14, 2H), 1.42 (m, J=9.51, 2H); MS m/z 295.9 (M+H)⁺.

Example 97 (6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)(piperidin-1-yl)methanone

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (50 mg, 0.205 mmol, 1 eq) and (6-chloropyrazin-2-yl)(piperidin-1-yl)methanone (75) (41 mg, 0.184 mmol, 0.9 eq) in DME (8 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (133 mg, 0.410 mmol, 2 eq) followed by addition of Pd(dpp)Cl₂ (6 mg, 0.00819 mmol, 0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 90° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1% MeOH/CHCl₃ to obtain pale yellow solid compound (6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)(piperidin-1-yl)methanone 138 in 10 mg quantity. The compound 138 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 9.61 (s, 1H), 9.12 (s, 1H), 9.02 (s, 1H), 8.80 (m, J=87.55 1H), 8.60 (d, J=1.82, 1H), 7.42 (m, J=3.17, 1H), 6.64 (m, J=1.58, 1H), 3.87 (s, 2H), 3.59 (m, J=88.29, 1H), 1.75 (s, 4H), 1.69 (m, J=24.99, 2H); MS m/z 307.9 (M+H)⁺.

Example 98 (6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)(4-methylpiperazin-1-yl)methanone

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (70 mg, 0.287 mmol, 1 eq) and (6-chloropyrazin-2-yl)(4-methylpiperazin-1-yl)methanone (60) (69 mg, 0.287 mmol, 1.0 eq) in DME (5 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (140 mg, 0.430 mmol, 1.5 eq) followed by addition of Pd(dpp)Cl₂ (13.2 mg, 0.0114 mmol, 0.04 eq) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 90° C. for 12 h in a sealed tube. After completion of the reaction, the reaction mixture was diluted with CHCl₃ and filtered through Celite. The solvents were distilled off and the crude material was submitted for flash column purification in neutral alumina using 1-2% MeOH/CHCl₃ to obtain pale yellow solid compound (6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)(4-methylpiperazin-1-yl)methanone 139 in 20 mg quantity. The compound 139 was confirmed by ¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 11.98 (s, 1H), 9.45 (s, 1H), 9.04 (d, J=1.95, 1H), 8.76 (m, J=12.43, 2H), 7.63 (m, J=2.68, 1H), 6.64 (m, J=1.58, 1H), 3.76 (s, 2H), 3.59 (m, J=69.03, 2H), 2.42 (m, 4H), 2.28 (s, 3H); MS m/z 321.4 (M+H)⁺.

Example 99 N-(1-methylpiperidin-4-yl)-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-amine

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (100 mg, 0.410 mmol) and 6-bromo-N-(1-methylpiperidin-4-yl)pyrazin-2-amine (11) (83 mg, 0.410 mmol) in DMSO (5 mL) was degassed and purged with nitrogen for 10 min. To this reaction mixture was added Cs₂CO₃ (226 mg, 2.0 eq) and the reaction mixture was purged again for another 10 min followed by the addition of Pd(PPh₃)₄ (0.03 eq). The resulting reaction mixture was heated at 80° C., overnight under sealed tube condition. After completion of the reaction the reaction mass was diluted with chloroform and filtered through Celite and the solvents were removed to obtain the crude product. The crude was passed through neutral alumina eluting with 2-3% MeOH/CHCl₃ providing beige coloured solid (12 mg) compound N-(1-methylpiperidin-4-yl)-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-amine 140. ¹H NMR (400 MHz, CDCl₃) δ: 8.94 (d, J=2.07, 1H), 8.80 (s, 1H), 8.50 (s, 1H), 8.32 (s, 1H), 7.81 (s, 1H), 7.35 (d, J=3.2 HZ, 1H), 6.59 (m, 1H), 2.89 (m, 2H), 2.24 (s, 3H), 2.17 (m, 4H), 1.25 m, 1H); MS m/z 309.1 (M+H)⁺.

Example 100 6-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyrazin-2-amine

Compound N-(1-methylpiperidin-4-yl)-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-amine (140).

Example 101 6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyrazin-2-amine

Example 102 N-methyl-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (124) (100 mg, 0.409 mmol) and 6-chloro-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (69) (90 mg, 0.409 mmol) in toluene (8 mL) and ethanol (2 ml) was degassed and purged under nitrogen for 10 min followed by the addition of Cs₂CO3 (260 mg, 0.819 mmol, 2.0 eq) and Pd(PPh3)4 (14 mg, 0.022 mmol, 0.03 eq) and the reaction mixture was degassed and purged again for 15 min. The resulting reaction contents were heated at 80° C. overnight under sealed condition. After completion of the reaction the mixture was diluted with chloroform and filtered through Celite and the organic layer was distilled off to get the crude product. The crude was passed through neutral alumina eluting with 1-2% MeOH/CHCl₃ to give off white colored solid N-methyl-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine 143 in 9 mg quantity. ¹H NMR (400 MHz, CDCl₃) δ: 9.05 (s, 1H), 8.97 (d, J=1.95, 1H), 8.53 (d, J=1.95, 1H), 8.34 (s, 1H), 7.94 (s, 1H), 7.36 (s, 1H), 6.60 (d, J=3.29, 1H), 4.01 (s, 2H), 3.56 (d, 2H), 3.21 (s, 3H), 3.01 (m, 4H), 2.24 (m, 2H), 2.22 (s, 1H); MS m/z 324.1 (M+H)⁺.

Example 103 6-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine

To a solution of compound N-methyl-6-(1H-pyrrolo[2,3-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazine-2-amine (143) (20 mg, 1.0 eq) in dichloromethane (5 mL) was added n-chlorosuccinamide (2.0 eq) and the resulting reaction mixture was stirred at room temperature overnight. After completion of the reaction the solvents were removed and extracted into CH₂Cl₂. The organic layer was separated and the aqueous phase was extracted with CH₂Cl₂ to retain the crude material. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed by rotary evaporator. The crude material was purified by flash column with 100-200 mesh silica gel employing 40% EtOAc/hexane eluents to obtain the pure off-white coloured solid of 6-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine compound 144.

Example 104 6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine

Fluorination of 6-(1H-pyrrolo[2,3-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (143), carried out using the reported procedures published in Organic Letters, 2011, 13(18), 4498-4501, incorporated herein by reference in its entirety, provided the title compound 6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine 145.

Example 105 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine

To a stirred solution of 5-bromo-1H-pyrrolo[2,3-b]pyridine (146) (1 g, 1 eq) in DMF, potassium acetate (6 eq), followed by bis-(pinacolato-diborane) (2 eq), was added. The reaction mixture was degassed with argon for 30 min. Then bis-(triphenylphosphine)-palladium (II) chloride (0.1 eq) was added and the reaction mixture was again degassed with argon for 30 minutes. Then the reaction mixture was heated to 90° C., stirred for 6 hr. Reaction mixture was concentrated under reduced pressure and the obtained residue was diluted with water and extracted with ethyl acetate (3×50 ml). The combined extract was washed with water, dried over anhydrous sodium sulphate, filtered and concentrated. Purification was done by flash column using 230-400 mesh silica gel and 20% ethyl acetate/petroleum ether to afford the desired product 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine 147 in 70% yields as white solid.

Example 106 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine

A stirred solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (147) (100 mg, 0.4 mmol) and 4-(6-chloropyrazin-2-yl)morpholine (103) (80 mg, 0.4 mmol) in DMF (5 mL) was degassed and purged with nitrogen for 10 min. To this reaction mixture was added Cs₂CO₃ (333 mg, 1.0 mmol, 2.5 eq) and the reaction mixture was purged again for another 10 min followed by the addition of Pd(PPh₃)₄ (0.018 mg, 0.016 mmol, 0.04 eq). The resulting reaction mixture was heated to 100° C., overnight under sealed tube condition. After completion of the reaction the mass was diluted with chloroform and filtered through Celite and the solvents were removed to obtain the crude product. The crude was passed through neutral alumina eluting with 2-3% MeOH/CHCl₃ provided a white coloured solid 25 mg of 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine 148. ¹H NMR (400 MHz, CDCl₃) δ: 8.96 (s, 1H), 8.95 (s, 1H), 8.84 (s, 1H), 8.54 (d, J=1.95, 1H), 8.43 (s, 1H), 8.07 (s, H), 7.36 (m, 1H), 3.71 (t, 4H), 3.69 (bt, 4H); MS m/z 281.90 (M+H)⁺.

Example 107 4-(6-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine

To a solution of compound 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine (148) (40 mg, 0.142 mmol, 1.0 eq) in dichloromethane (6 mL) was added n-chlorosuccinamide (38 mg, 0.283 mmol, 2.0 eq) and the resulting reaction mixture was stirred at room temperature overnight. After completion of the reaction the solvents were removed and extracted into CH₂Cl₂. The organic layer was separated and the aqueous phase was extracted with CH₂Cl₂ to retain the crude material. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed by rotary evaporator. The crude material was purified by flash column with 100-200 mesh silica gel employing 70% EtOAc/hexane eluents to obtain the pure off-white colored solid of 4-(6-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine 149 (10 mg). ¹H NMR (400 MHz, CDCl₃) δ: 9.0 (s, 1H), 8.83 (s, 1H), 8.52 (s, 1H), 8.45 (s, 1H), 8.10 (s, 1H), 7.34 (d, J=2.31 Hz, 1H), 3.89 (m, 4H), 3.70 (m, 4H); MS m/z 315.9 (M+H)⁺.

Example 108 4-(6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine

Fluorination of 4-(6-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine (148) carried out using the reported procedures published in Organic Letters, 2011, 13(18), 4498-4501, incorporated herein by reference in its entirety, provided the title compound 4-(6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine 150 in 6 mg quantity.

Example 109 1-(4-methoxybenzyl)-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine

A stirred solution of 1-(4-methoxybenzyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (147PG) (50 mg, 0.137 mmol) and 1-(4-bromobenzyl)-4-methylpiperazine (121) (37 mg, 0.137 mmol, 1.1 eq) in DME (10 mL) was degassed and purged with N₂ for 10 min and Cs₂CO₃ (2.0 eq) was added and the reaction mixture purged and degassed again. To this reaction mixture was added Pd(PPh3)₄ (0.04 eq) and the resulting reaction was heated at 90° C. for 12 hrs in sealed tube condition. After completion of the reaction the contents were was cooled and diluted with CH₂Cl₂ and filtered through Celite bed. The CH₂Cl₂ layer was completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound 1-(4-methoxybenzyl)-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine 151 obtained with 5% MeOH in CH₂Cl₂ as an off-white coloured solid in 30 mg quantity.

Example 110 5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine

To a stirred solution of 1-(4-methoxybenzyl)-5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (151) (30 mg) in CHCl₃ (8 mL) was added trifluoroacetic acid (5 mL) and heated at 50° C. for 12. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with CHCl₃ twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound in 2% MeOH/CHCl₃ gave pale yellow solid compound 5-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine 152.

Example 111 N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acrylamide

The starting material 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (134 mg, 0.402 mmol, 1 eq) and (3-acrylamidophenyl)boronic acid (153) (100 mg, 0.366 mmol, 1 eq) in DME (10 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (233 mg, 0.732 mmol, 2 eq) followed by addition of Pd(PPh₃)₄ (0.016 mg, 0.03 mmol) and degassing and purging under argon for additional 10 min. The reaction mixture was heated at 100° C. for 16 h in a sealed tube. After completion of the reaction, the solvents were removed and reaction mixtures were extracted into EtOAc, dried over Na₂SO₄ and the solvents distilled off. The crude material was submitted for flash column purification using 40% EtOAc in hexane to obtain off-white solid compound N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acrylamide 154. MS m/z 397.2 (M+H)⁺.

Example 112 N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acrylamide

To a stirred solution of N-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acrylamide (154) (70 mg) in CHCl₃ (20 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture heated at 50° C. for 12. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound in 60% EtOAc/hexane gave pale yellow solid compound N-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acrylamide 155. ¹H NMR (400 MHz, CDCl₃) δ: 10.29 (s, H), 8.75 (d, J=2.07, 1H), 8.40 (d, J=1.95, 1H), 8.03 (s, 1H), 7.69 (s, 1H), 7.43 (m, 1H), 6.50 (m, 1H), 6.46 (m, 1H), 6.26 (m, 1H), 2.55 (bs, 3H); MS m/z 277.9 (M+H)⁺.

Example 113 2-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetonitrile

The starting material 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (136 mg, 0.41 mmol, 1 eq) and 2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetonitrile (156) (100 mg, 0.41 mmol, 1 eq) in DME (10 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (260 mg, 0.82 mmol, 2 eq) followed by addition of Pd(PPh₃)₄ (15 mg, 0.03 mmol), degassing and purging under argon for additional 10 min. The reaction mixture was heated at 80° C. for 12 h in a sealed tube. After completion of the reaction, the solvents were removed and reaction mixtures were extracted into EtOAc, dried over Na2SO4 and the solvents distilled off. The crude material was submitted for flash column purification using 20% EtOAc in hexane to obtain off-white solid compound 2-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetonitrile 157.

Example 114 2-(3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetonitrile

To a stirred solution of 2-(3-(1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)acetonitrile (157) (60 mg) in CHCl₃ (20 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was heated at 50° C. for 12. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound in 50% EtOAc/hexane gave pale yellow solid compound 158 in 20 mg quantity.

Example 115 1-(4-methoxybenzyl)-3-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridine

The starting material 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (145 mg, 0.44 mmol, 1 eq) and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine (159) (135 mg, 0.44 mmol, 1 eq) in DME (10 mL) was degassed and purged under argon atmosphere for 10 min. To this reaction mixture was charged Cs₂CO₃ (280 mg, 0.88 mmol, 2 eq) followed by addition of Pd(PPh₃)₄ (15 mg, 0.01 mmol), degassing and purging under argon for additional 10 min. The reaction mixture was heated at 80° C. for 12 h in a sealed tube. After completion of the reaction, the solvents were removed and reaction mixtures were extracted into EtOAc, dried over Na2SO4 and the solvents distilled off. The crude material was submitted for flash column purification using 5% MeOH/CHCl₃ to obtain off-white solid compound 1-(4-methoxybenzyl)-3-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridine 160 in 65 mg quantity.

Example 116 3-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridine

To a stirred solution of 1-(4-methoxybenzyl)-3-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridine (160) in CHCl₃ (20 mL) was added trifluoroacetic acid (5 mL) and heated at 50° C. for 12 hours. After completion of the reaction the solvents were removed, diluted with cold water, pH was adjusted to 8 and the aqueous phase extracted with chloroform twice. The organic layer was washed with brine solution and dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound in 10% MeOH/CHCl₃ as pale yellow solid compound 161 (8 mg).

Example 117 2-(4-(3-((1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)amino)benzyl)piperazin-1-yl)ethanol

A stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (80 mg, 0.241 mmol, 1.0 eq) and 2-(4-(3-aminobenzyl)piperazin-1-yl)ethanolin (162) (56.7 mg, 0.241 mmol) 1,4-dioxane (8 mL) was degassed and purged with N₂ for 10 min. tert-BuOK (3.0 eq) was added and the reaction mixture was purged and degassed again. To this reaction mixture was added ±BINAP (8 mg, 0.00301 mmol, 0.01 eq) and Pd₂(dba)₃ (0.04 eq) and the resulting reaction was heated at 90° C. for 12 hrs in sealed tube condition. After completion of the reaction the contents were cooled and diluted with CHCl₃ and filtered through Celite bed. The CHCl₃ layer was completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound 2-(4-(3-((1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)amino)benzyl)piperazin-1-yl)ethanol 163 obtained with 6% MeOH in CH₂Cl₂ as an off-white colored solid in 9 mg quantity.

Example 118 2-(4-(3-((3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)amino)benzyl)piperazin-1-yl)ethanol

To a stirred solution of 2-(4-(3-((1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)amino)benzyl)piperazin-1-yl)ethanol (163) (50 mg, 0.103 mmol) in CHCl₃ (10 mL) was added trifluoroacetic acid (6 mL) and the reaction mass heated at 60° C. overnight. After completion of the reaction the solvents were completely distilled off and diluted with water and the pH was adjusted to 9 and extracted with chloroform twice. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed and the crude material was passed through 100-200 mesh silica gel, eluting with 10% MeOH/CH₂Cl₂ obtained pure 2-(4-(3-((3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)amino)benzyl)piperazin-1-yl)ethanol 164 as a pale yellow colored solid (6 mg). MS m/z 353.54 (M+H)⁺.

Example 119 1-(4-methoxybenzyl)-3-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-5-amine

A stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (50 mg, 0.150 mmol) and 4-((4-methylpiperazin-1-yl)methyl)aniline (165) (30 mg, 0.165 mmol, 1.1 eq) in 1,4-dioxane (10 mL) was degassed and purged with N₂ for 10 min. tert-BuOK (50 mg, 0.451 mmol, 3.0 eq) was added and the reaction mixture was purged and degassed again. To this reaction mixture was added ±BINAP (1.8 mg, 0.00301 mmol, 0.01 eq) and Pd₂(dba)₃ (5 mg, 0.0012 mmol, 0.04 eq) and the resulting reaction was heated at 90° C. for 12 hrs in sealed tube condition. After completion of the reaction the contents were cooled and diluted with CHCl₃ and filtered through Celite bed. The CHCl₃ layer was completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound 1-(4-methoxybenzyl)-3-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-5-amine 166 obtained with 6% MeOH in CH₂Cl₂ as an off-white coloured solid in 30 mg quantity.

Example 120 3-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-5-amine

To a stirred solution of 1-(4-methoxybenzyl)-3-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-5-amine (166) (40 mg, 0.657 mmol) in CHCl₃ (10 mL) was added trifluoroacetic acid (6 mL) and the reaction mass heated at 60° C. overnight. After completion of the reaction the solvents were completely distilled off and diluted with water and the pH was adjusted to 9 and extracted with chloroform twice. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed and the crude material was passed through 100-200 mesh silica gel, eluting with 8% MeOH/CH₂Cl₂ obtained pure 3-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-5-amine 167 as a pale yellow colored solid (10 mg). ¹H NMR (400 MHz, CDCl₃) δ: 8.38 (s, 1H), 7.76 (s, 1H), 7.52 (s, 1H), 7.21 (d, 1H), 7.19 (d, J=8.41, 1H), 7.19 (s, 1H), 6.89 (d, J=7.92, 1H), 3.46 (s, 2H), 2.54 (m, 8H), 2.31 (s, 3H), 2.05 (s, 3H); MS m/z 336.9 (M+H)⁺.

Example 121 N-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine

A stirred solution of 5-bromo-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridine (7) (100 mg, 0.301 mmol) and 3-fluoro-4-(4-methylpiperazin-1-yl)aniline (168) (77 mg, 0.33 mmol, 1.1 eq) in 1,4-dioxane (10 mL) was degassed and purged with N₂ for 10 min. tert-BuOK (101 mg, 0.903 mmol, 3.0 eq) was added and the reaction mixture purged and degassed again. To this reaction mixture was added ±BINAP (8 mg, 0.00301 mmol, 0.01 eq) and Pd₂(dba)₃ (11 mg, 0.0012 mmol, 0.04 eq) and the resulting reaction was heated at 90° C. for 12 hrs in sealed tube condition. After completion of the reaction the contents were cooled and diluted with CHCl₃ and filtered through Celite bed. The CHCl₃ layer was completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel, eluting the pure compound N-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine 169 obtained with 6% MeOH in CH₂Cl₂ as an off-white colored solid in 40 mg quantity.

Example 122 N-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine

To a stirred solution of N-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-1-(4-methoxybenzyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine (169) (30 mg, 0.063 mmol) in CHCl₃ (15 mL) was added trifluoroacetic acid (6 mL) and the reaction mass heated to 60° C. overnight. After completion of the reaction the solvents were completely distilled off and diluted with water and the pH was adjusted to 9 and extracted with chloroform twice. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed and the crude material was passed through 100-200 mesh silica gel, eluting with 8% MeOH/CH₂Cl₂ obtained pure N-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine 170 as a pale yellow colored solid (10 mg). ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (s, 1H), 7.71 (s, J=2.31, 1H), 6.90 (m, 1H), 6.71 (m, 2H), 5.56 (s, 1H), 3.07 (bs, 4H), 2.62 (bs, 4H), 2.54 (bs, 3H), 2.36 (s, 3H); MS m/z 341.0 (M+H)⁺.

Example 123 N-(6-chloropyrazin-2-yl)-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-amine

To a stirred solution of 5-bromo-3-methyl-1H-pyrazolo[3,4-b]pyridin (6) and 2,6-dichloropyrazine (52) in tert-butanol which was degassed and purged with nitrogen was added tert-BuOK, Pd₂(dba)₃ and s-phos and the reaction materials were again degassed, purged with nitrogen for 15 min. The reaction mass was heated at 90° C. for 14 hrs under sealed condition. After completion of the reaction the RM was diluted with water and extracted with dichloromethane twice. The combined organic layer was dried over sodium sulphate and the solvent completely distilled off to get the crude product. The crude was passed through 100-200 mesh silica gel eluting the pure compound at 30% EtOAc in hexane as brown colour solid 171 (50 mg). ¹H NMR (400 MHz, CDCl₃) δ: 9.52 (s, 1H), 8.55 (s, 1H), 8.41 (d, J=2.56, 1H), 7.00 (s, 1H), 3.77 (s, 3H).

TABLE 1 List of Compounds Exp. ID Structure Chemical Name Mol. Wt  13

6-(3methyl-1H-pyrazolo [3,4- b]pyridine-5yl)-N-(1-methylpiperidine- 4yl) pyrazin-2-amine 323.40  13HCL

6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)-N-(1-methylpiperidin- 4-yl)pyrazin-2-amine hydrochloride 359.51  19

5-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)-N-(1-methylpiperidin- 4-yl)pyrimidin-4-amine 323.40  22

N-(1-(4-fluorobenzyl)piperidin-3-yl)-3- methyl-1H-pyrazolo[3,4-b]pyridin-5- amine 339.41  26

N-(1-(3,4-difluorobenzylpiperidin-3- yl)-3-methyl-1H-pyrazolo[3,4- b]pyridid-5-amine 357.40  27

N-(1-(4-chlorobenzyl)piperidin-3-yl)- 3-methyl-1H-pyrazolo[3,4-b]pyridin-5- amine 355.86  28

N-(1-(3,4-difluorobcnzyl)piperidin-3- yl)-3-methyl-1H-pyrazolo[3,4- b]pyridin-5-amine 390.31  33

3-methyl-5-(6-(2-(4-methylpiperazin-1- yl)ethoxy)pyrazin-2-yl)-1H- pyrazolo[3,4-b]pyridine 353.20  39

1-methyl-N-(6-(3-methyl-1H- pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2- yl)piperidine-4-carboxamide 351.41  42

5-(6-chloropyrazin-2-yl)-3-methyl-1H- pyrazolo[3,4-b]pyridine 245.67  45

3-methyl-5-(pyrazin-2-yl)-1H- pyrazolo[3,4-b]pyridine 211.22  48

1-(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)pyrrolidin- 2-one 294.31  51

6-(3-mcthyl-1H-pyrazolo[3,4- b]pyridin-5-yl)-N-(1-methylpyrrolidin- 3-yl)pyrazin-2-amine 309.37  56

5-(6-(3,3-difluoropyrrolidin-1- yl)pyrazin-2-yl)-3-methyl-1H- pyrazolo[3,4-b]pyridine 316.31  59

2-(4-(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)piperazin- 1-yl)ethanol 339.39  62

(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)(4- methylpiperazin-1-yl)methanone 337.38  62

(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)(piperazin- 1-yl)methanone 323.35  65

3-methyl-5-(6-(4-methylpiperazin-1- yl)pyrazin-2-yl)-1H-pyrazolo[3,4- b]pyridine 309.37  68

N1,N1-dimethyl-N2-(6-(3-methyl-1H- pyrazolo[3,4-b]pyridin-5-yl)pyrazin-2- yl)ethane-1,2-diamine 297.36  71

N-methyl-6-(3-methyl-1H- pyrazolo[3,4-b]pyridin-5-yl)-N- ((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine 338.41  74

3-methyl-5-(3-(pyrrolidin-1-yl)pyrazin- 2-yl)-1H-pyrazolo[3,4-b]pyridine 280.33  77

(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)(piperidin- 1-yl)methanone 322.36  81

2-(4-((6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2- yl)amino)piperidin-1-yl)ethanol 353.42  83

4-(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)piperazin- 2-one 309.33  87

N-(1-isopropylpiperidin-4-yl)-6-(3- methyl-1H-pyrazolo[3,4-b]pyridin-5- yl)pyrazin-2-amine 351.45  90

5-(6-(indolin-1-yl)pyrazin-2-yl)-3- methyl-1H-pyrazolo[3,4-b]pyridine 328.37  94

1-(3-((6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2- yl)amino)phenyl)pyrrolidin-2-one 385.42  98

1-methyl-N-(3-(3-methyl-1H- pyrazolo[3,4-b]pyridin-5- yl)phenyl)piperidin-4-amine 321.42 102

6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)-N-(1-methylpiperidin- 4-yl)pyridin-2-amine 322.41 105

4-(6-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)pyrazin-2-yl)morpholine 296.33 108

3-methyl-5-(1-methyl-1H-pyrazol-4- yl)-1H-pyrazolo[3,4-b]pyridine 213.24 111

N,N-dimethyl-3-(3-methyl-1H- pyrazolo[3,4-b]pyridin-5- yl)benzenesulfonamide 316.38 114

N-(3-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)phenyl)acetamide 266.30 117

5-(1-methyl-1H-pyrazol-4-yl)-1H- pyrazolo[3,4-b]pyridine 199.21 119

ethyl 3-(3-mcthyl-1H-pyrazolo[3,4- b]pyridin-5-yl)benzoate 281.31 123

3-methyl-5-(3-((4-methylpiperazin-1- yl)methyl)phenyl)-1H-pyrazolo[3,4- b]pyridine 321.42 125

5-(6-(4-methylpiperazin-1-yl)pyrazin- 2-yl)-1H-pyrrolo[2,3-b]pyridine 429.35 126

2-(4-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)piperazin-1-yl)ethanol 324.38 127

N1-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)-N2,N2- dimethylethane-1,2-diamine 282.34 128

5-(3-(pyrrolidin-1-yl)pyrazin-2-yl)-1H- pyrrolo[2,3-b]pyridine 265.31 130

N-(1-methylazetidin-3-yl)-6-(1H- pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2- amine 280.33 131

1-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)pyrrolidin-2-one 279.30 133

4-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)piperazin-2-one 294.31 134

5-(6-(indolin-1-yl)pyrazin-2-yl)-1H- pyrrolo[2,3-b]pyridine 313.36 135

5-(6-(3,3-difluoropyrrolidin-1- yl)pyrazin-2-yl)-1H-pyrrolo[2,3- b]pyridine 301.29 137

1-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)piperidin-4-ol 295.34 138

(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)(piperidin-1- yl)methanone 307.35 139

(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)(4-methylpiperazin-1- yl)methanone 322.36 140

N-(1-methylpiperidin-4-yl)-6-(1H- pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2- amine 308.38 141

6-(3-chloro-1H-pyrrolo[2,3-b]pyridin- 5-yl)-N-(1-methylpiperidin-4- yl)pyrazin-2-amine 342.83 142

6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin- 5-yl)-N-(1-methylpiperidin-4- yl)pyrazin-2-amine 326.37 143

N-methyl-6-(1H-pyrrolo[2,3-b]pyridin- 5-yl)-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine 323.39 144

6-(3-chloro-1H-pyrrolo[2,3-b]pyridin- 5-yl)-N-methyl-N-((tetrahydro-2H- pyran-4-yl)methyl)pyrazin-2-amine 357.84 145

6-(3-fluoro-1H-pyrrolo[2,3-b]pyridin- 5-yl)-N-methyl-N-((tetrahydro-2H- pyran-4-yl)methyl)pyrazin-2-amine 341.38 148

4-(6-(1H-pyrrolo[2,3-b]pyridin-5- yl)pyrazin-2-yl)morpholine 281.31 149

4-(6-(3-chloro-1H-pyrrolo[2,3- b]pyridin-5-yl)pyrazin-2-yl)morpholine 315.76 150

4-(6-(3-fluoro-1H-pyrrolo[2,3- b]pyridin-5-yl)pyrazin-2-yl)morpholine 299.30 152

5-(3-((4-methylpiperazin-1- yl)methyl)phenyl)-1H-pyrrolo[2,3- b]pyridine 306.40 155

N-(3-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)phenyl)acrylamide 278.31 158

2-(3-(3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)phenyl)acetonitrile 248.28 161

3-methyl-5-(4-(4-methylpiperazin-1- yl)phenyl)-1H-pyrazolo[3,4-b]pyridine 307.39 164

2-(4-(3-((3-methyl-1H-pyrazolo[3,4- b]pyridin-5-yl)amino)benzyl)piperazin- 1-yl)ethanol 366.46 167

3-methyl-N-(4-((4-methylpiperazin-1- yl)methyl)phenyl)-1H-pyrazolo[3,4- b]pyridin-5-amine 336.43 170

N-(3-fluoro-4-(4-methylpiperazin-1- yl)phenyl)-3-methyl-1H-pyrazolo[3,4- b]pyridin-5-amine 340.40 171

N-(6-chloropyrazin-2-yl)-3-methyl-1H- pyrazolo[3,4-b]pyridin-5-amine 260.68 General Procedures for the Preparation Compounds 173-182

The key compound 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine 172 protected with either triphenylmethyl chloride (trityl chloride), p-toluenesulfonyl chloride (p-TsCl) and/or tert-Butyloxycarbonyl (BOC) group (1 eq) was reacted with 2-substituted heteroaryl halogen containing scaffolds; 11, 46, 57, 63, 69, 72, 88, 103, 109 and 129 (0.9 to 1.0 eq) in either DMF, DME, Toluene, Acetonitrile, tert-BuOH, THF and or 1,4-dioxane were taken in a sealed tube. The resulting reaction mixture was degassed, purged with argon or N₂ gas a few times and was charged with NaCO₃, Cs₂CO₃, K₂CO₃, potassium acetate or NaHCO₃ (1.5 to 2 equivalents) followed by the addition of palladium catalysts (0.01 to 0.05 eq). After the addition of catalysts the contents of the reaction were purged and degassed again and heated at 80 to 100° C. for 8 to 16 hrs. After completion of the reaction monitored from TL, the contents were cooled to room temperature and diluted with CH₂Cl₂, CHCl₃ or EtOAc. The organic layers were passed through a Celite pad then the solvent was completely distilled off to get the crude product. The crude was subjected to flash to column chromatography purification to get the title compounds.

General Procedures for the Deprotection to Give Compounds 173-182

Deprotection reactions were carried out (unless specified otherwise) in CH₂Cl₃, CHCl₃ or 1,4-dioxane (5-10 mL) and trifluoroacetic acid (5 mL) or concentrated HCl. The reaction mixture was stirred at RT or heated at 40° to 50° C. for 4 to 10 hrs. After completion of the reaction the solvents were completely distilled off and diluted with water and the pH was adjusted to 9 and extracted with chloroform twice. The combined organic layer was washed with brine solution and dried over sodium sulphate and the solvents were removed and the crude material was passed through 100-200 mesh silica gel, eluting with 1 to 10% MeOH/CH₂Cl₂ or CHCl₃ to obtain off-white to pale yellow coloured compounds 173-182.

Example 124 6-(3-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-(1-methylpiperidin-4-yl)pyrazin-2-amine (173)

¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 8.91 (d, J=1.82 Hz 1H), 8.55 (s, 1H), 8.42 (d, J=1.70 Hz 1H), 8.33 (s, J=34.04 Hz 1H), 7.81 (s, 1H), 7.10 (s, 1H), 4.57 (d, J=8.29 Hz 1H), 3.85 (s, 1H), 2.86 (s, 2H), 2.34 (m, J=13.65 Hz 6H), 2.18 (s, 4H), 1.68 (s, 2H), 1.25 (s, 2H); MS m/z 322.9 (M+H)⁺.

Example 125 N-methyl-6-(3-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (174)

¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 8.96 (d, J=1.82 Hz 1H), 8.45 (d, J=1.70 Hz 2H), 8.35 (d, J=40.85 Hz 1H), 7.94 (s, 1H), 7.10 (s, 1H), 3.99 (m, J=7.43 Hz 2H), 3.55 (m, J=7.19 Hz 2H), 3.38 (m, J=1.82 Hz 2H), 3.22 (s, 3H), 2.37 (s, 3H), 1.68 (m, J=13.29 2H), 1.46 (m, J=7.31 Hz 2H); MS m/z 337.9 (M+H)⁺.

Example 126 4-(6-(3-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazin-2-yl)morpholine (175)

¹HNMR and LCMS. ¹H NMR (400 MHz, CDCl₃) δ: 8.93 (d, J=1.95 Hz 1H), 8.57 (s, 1H), 8.44 (s, 1H), 8.07 (s, 1H), 7.11 (s, 1H), 3.88 (m, J=5.12 Hz 4H), 3.69 (m, J=5.12 Hz 4H), 2.38 (s, 3H); MS m/z 259.9 (M+H)⁺.

TABLE 2 List of additional compounds 83

3-(2-methoxyphenyl)-5-(thiophen-3-yl)- 1H-pyrazolo[3,4-b]pyridine MS m/z 307.37 (M + H)⁺. 84

5-(furan-3-yl)-3-(2-methoxyphenyl)- 1H-pyrazolo[3,4-b]pyridine MS m/z 291.30 (M + H)⁺. Pharmaceutical Salts

Hydrochloric acid salts of the foregoing compounds were prepared by solubilizing the compound in a minimum of ethanol and a solution of ethanolic HCl 20% was added drop wise and the mixture stirred for 1 hour followed by addition of diethyl ether. A precipitated off-white solid hydrochloride was separated by filtration, washer with diethyl ether and dried.

Additional Examples

Usage Method

The subject matter disclosed herein relates to the substituted 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine and pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors. Pharmaceutical compositions containing the Formula I, II and VII compounds and methods of using the compounds or compositions to treat various types of diseases or conditions such as Parkinson's disease, Alzheimer's disease, Down's syndrome, Huntington's disease, other neurodegenerative and or CNS, cancer, metabolic and inflammatory disease patients are disclosed. Additionally these compounds and compositions containing the compounds may be employed to treat pain of neuropathic origin, pain of inflammatory origin, cardiovascular disease, rheumatoid arthritis, osteoarthritis, type 2 diabetes, metabolic syndrome and obesity. Methods of making the compounds and pharmaceutical salts thereof are also described herein.

The in vivo neuroprotective efficacy of 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine and pyrazolo[3,4-b]pyridine in an established MPTP model of PD is described. The C57 black mouse has been used since it is the most sensitive strain for MPTP-induced dopaminergic neurotoxicity model. Sub-chronic MPTP dosing is performed with multiple doses spread out over a 5 day period. MPTP at a dose of 20-25 mg/kg (i.p.) once a day for 5 days results in over 80% depletion of striatal dopamine and significant neuronal damage seven days after MPTP treatment. Importantly, the sub-chronic model produces substantial apoptotic cell death, without overt toxicity, and significant necrotic cell death. PD and similar models were tested for post-treatment studies. The series of compounds was administered after significant depletion of striatal dopamine.

In Vitro Inhibition Assay

LRRK2 Kinase Assay

Procedure: Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μl of 3% phosphoric acid. Volume was increased to 100 μl by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μl 0.8% phosphoric acid and dried for an hour at 60° C. 100 μl scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values. A validation EC₅₀ curve was generated by plotting CPM for each Staurosporine concentration on y-axis against the Log concentration of Staurosporine (nM) on the x-axis followed by a best fit curve through the points.

% Inhibition=((Enzyme Control−Compound Treated)/Enzyme Control)×100 Coefficient of Variance (% CV) between replicates: The % CV values between the replicates were mostly within the acceptable limits of a radiometric experiment. Z′ factor evaluation: The value of Z′ factor was found to be 0.8 for LRRK2 (SEQ ID NO 62) WT and 0.9 was derived for LRRK2 G2019S (SEQ ID NO 28).

All the compounds were tested in 10-dose IC₅₀ mode with 3 fold serial dilution starting at 100 μM. The control compound Staurosporine was tested in 10 dose IC50 with 3 fold serial dilution starting at 20 μM. The reactions were carried out at 10 μM ATP for LRRK2 (SEQ ID NO 62) and LRRK2 G2019 (SEQ ID NO 28)(FIG. 1).

General Protein Kinase Assay Methodology Employed for Selected Kinases

In vitro profiling of the 317 member kinase panel was performed at Reaction Biology Corporation using the “HotSpot” assay platform. Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed ˜20 min later by addition of a mixture of ATP (Sigma) and ₃₃P ATP (PerkinElmer) to a final concentration of 10 μM. Reactions were carried out at 25° C. for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC₅₀ values and curve fits were obtained using Prism (GraphPad Software). Kinome tree representations were prepared using Kinome Mapper (http://www.reactionbiology.com/apps/kinome/mapper/Launch Kinome.htm). Specifically for the LRRK2 enzyme the substrate LRRKtide (gene bank and protein access numbers NP_(—)940980.2 Q5S007) and clone aa 970-2527 expressed in baculovirus in insect cells with N-terminal GST tag was employed in the study protocol. In the case of DYRK1A (SEQ ID NO 16) the substrate casein (gene bank and protein access numbers NP_(—)001387 Q13627) full length clone expressed in insect with n-terminal tag was used.

Adapta Screening Assay Conditions

The ADP Transcreener uses direct immunodetection of ADP with three different fluorescent detection modes (FP, TR-FRET and FI) and provides the most sensitive HTS method for kinases/ATP-utilizing enzymes. The HTRF Transcreener ADP assay is based on the direct detection of ADP using anti-phospho LRRKtide antibody labelled with Eu³⁺ cryptate. LRRK2 undergoes weak autophosphorylation whereas it exhibits high activity towards the peptidic substrate LRRKtide. This assay can be used for high throughput screening or profiling of any ATP/ADP dependent target such as LRRK2.

For the wild type LRRK the 2×LRRK2/ERM (LRRKtide) mixture was prepared in 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction consisted of 3.5-20.8 ng LRRK2 and 200 μM ERM (LRRKtide) in 25 mM Tris/7.5 mM HEPES pH 8.2, 0.005% BRIJ-35, 5 mM MgCl2, 0.5 mM EGTA, 0.01% NaN3. After 1 hour Kinase Reaction incubation, 5 μL of Detection Mix was added.

In the case of LRRK2 G2019S the 2×LRRK2 G2019S/ERM (LRRKtide) mixture was prepared in 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction consisted of 2-10 ng LRRK2 G2019S and 200 μM ERM (LRRKtide) in 25 mM Tris/7.5 mM HEPES pH8.2, 0.005% BRIJ-35, 5 mM MgCl2, 0.5 mM EGTA, 0.01% NaN3. After 1 hour Kinase Reaction incubation, 5 μL of Detection Mix was added.

In Vitro DYRK1A, DYRK1B, DYRK2 and DYRK3 Inhibition Assay

The 2×DYRK1A/Ser/Thr 18 mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL Kinase Reaction consisted of 1.68-12.5 ng DYRK1A (SEQ ID NO 16) and 2 μM Ser/Thr 18 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A was added.

The 2×DYRK1B (SEQ ID NO 17)/Ser/Thr 18 mixture was prepared in 50 mM HEPES pH 6.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.02% NaN3. The final 10 μL Kinase Reaction consisted of 0.47-5.84 ng DYRK1B (SEQ ID NO 17) and 2 μM Ser/Thr 18 in 50 mM HEPES pH 7.0, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.01% NaN3. After 1 hour Kinase Reaction incubation, 5 μL of a 1:1024 dilution of Development Reagent A was added.

The 2×DYRK2 (SEQ ID NO 18)/Ser/Thr 09 mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL Kinase Reaction consisted of 1.92-10.7 ng DYRK2 (SEQ ID NO 18) and 2 μM Ser/Thr 09 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A is added.

The 2×DYRK3 (SEQ ID NO 19)/Ser/Thr 09 mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL Kinase Reaction consisted of 1.92-10.7 ng DYRK3 (SEQ ID NO 19) and 2 μM Ser/Thr 09 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent A is added.

TABLE 3 List of Compounds and Corresponding Protein Kinase Inhibition* LRRK2 Compound LRRK2 G2019S ID IC₅₀ μM IC₅₀ μM DYRK1A 13 *** *** *** 13 *** *** *** 19 ** ** ND 22 ** ** ** 26 ** ** ND 27 ** ** ** 28 ** ** ** 33 ** ** ** 39 ** ** ** 42 * * ND 45 * * ND 48 ** ** * 51 ** ** * 56 ** ** * 59 *** *** ** 62 ** ** ** 65 *** *** * 68 *** *** * 71 *** *** *** 74 * * ND 77 ** ** * 81 *** *** *** 83 ** ** ** 87 ** ** ** 90 *** *** *** 94 *** *** ** 98 * * * 102 ** ** ** 105 *** *** *** 108 ** ** ** 111 * * ND 114 * * ND 117 * * ND 119 * * ND 123 * * ND 125 *** *** *** 126 *** *** *** 127 ** ** ** 128 ** ** ** 130 ** ** ND 131 ** ** ** 133 ** ** ND 134 *** *** *** 135 ** ** ** 137 *** *** *** 138 ** ** ND 139 * * ND 140 *** *** ND 141 *** *** ND 142 *** *** *** 143 *** *** *** 144 *** *** *** 145 *** *** *** 148 *** *** *** 149 *** *** *** 150 *** *** *** 152 *** *** *** 155 * * ND 158 * * ND 161 * * ND 164 * * ND 167 * * ND 170 * * ND 171 * * ND 183 *** *** *** 184 *** *** *** *Kinase Inhibition Result for Selected Compounds *** <0.5 μM, ** >0.5 μM, * >1 μM ND = Not Determined Protein Kinase Selectivity Profiler

Selected LRRK2 (SEQ ID NO 62) and LRRK2 G2019S (SEQ ID NO 28) inhibiting compounds 13, 13 HCl, 71, 105, 134 and 148 were tested against 299 protein kinases+17 additional preview kinases in single dose duplicate mode at a concentration of 0.5 μM, 1 μM and 1 μM). A control compound was tested in 10-dose IC₅₀ mode with 3-fold serial dilution starting at 20 μM. Reactions were carried out at 10 μM ATP. Data pages include raw data, % Enzyme activity (relative to DMSO controls), and curve fits. The compounds 105, 134 and 148 are selective in inhibiting some kinases including LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), CHK1 (SEQ ID NO 8), DYRK1A (SEQ ID NO 16), FLT3 (SEQ ID NO 21), MLK1 (SEQ ID NO 33), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56) with % enzyme inhibition of 5-15% relative to DMSO controls, whereas the compounds 13 and 71 had activity against kinases in broader scope, inhibiting ABL1 T315I (SEQ ID NO 1), ALK2 (SEQ ID NO 2), ARK5 (SEQ ID NO 64), Aurora A (SEQ ID NO 3), Aurora B (SEQ ID NO 4), Aurora C (SEQ ID NO 5), BRAF (SEQ ID NO 6), BRAF V599E, C-KIT (SEQ ID NO 10), CLK1 (SEQ ID NO 11), CLK2 (SEQ ID NO 12), CLK3 (SEQ ID NO 13), CLK4 (SEQ ID NO 14), CSF1R (SEQ ID NO 15), DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17), DYRK 2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20), FLT3 (SEQ ID NO 21), GSK3β (SEQ ID NO 22), JAK1 (SEQ ID NO 23), JAK2 (SEQ ID NO 24), JAK3 (SEQ ID NO 25), KDR (SEQ ID NO 26), LCK (SEQ ID NO 27), MAP4K4 (SEQ ID NO 30), MELK (SEQ ID NO 31), MLK (SEQ ID NO 32), PDGFR (SEQ ID NO 41), PKA (SEQ ID NO 63), PKCα (SEQ ID NO 42), PKCτ (SEQ ID NO 43), PIM1 (SEQ ID NO 44), RET (SEQ ID NO 45), ROCK1 (SEQ ID NO 46), ROCK2 (SEQ ID NO 47), RSK1 (SEQ ID NO 48), RSK2 (SEQ ID NO 49), p70S6K (SEQ ID NO 50), SGK1 (SEQ ID NO 51), SNF1LK (SEQ ID NO 52), SIK2 (SEQ ID NO 53), SYK (SEQ ID NO 54), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56), TRKB (SEQ ID NO 57) or TRKC (SEQ ID NO 58) kinases in the range of 5 to 15% relative to DMSO control.

Cell Culture Models of Parkinsons Disease

Rat Mesencephalic Cell Line (N27 Cells):

N27 dopaminergic neuronal cell line has been widely used as a useful model for testing neuroprotective agents ((Kaul et al., 2003; Yang et al., 2004; Zhang et al., 2007). N27 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 50 units penicillin, and 50 μg/ml streptomycin (Kaul et al., 2003; Yang et al., 2004). The cell line was incubated at 37° C. in a humidified atmosphere containing 5% CO₂.

Primary Mesencephalic Neuronal Cultures:

Primary mesencephalic neuronal cultures were prepared from the ventral mesencephalon of gestational 16- to 18-day-old mouse embryos, as previously described (Zhang et al., 2007). Mesencephalic tissues were dissected from E16 to E18 mouse embryos maintained in ice cold Ca2+-free Hanks' balanced salt solution and then dissociated in Hanks' balanced salt solution containing trypsin-0.25% EDTA for 30 min at 37° C. The dissociated cells were then plated at equal density (0.5×106 cells) on 12 mm coverslips precoated with 0.1 mg/ml poly-D-lysine. Cultures were maintained in neurobasal medium fortified with B-27 supplements, 500 μM 1-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The cells were maintained in a humidified CO2 incubator (5% CO2 and 37° C.). Half of the culture medium was replaced every 2 days. Primary mesencephalic cultures (7-10 days old) were co-treated with 10 μM MPP+±test compound (1, 3 and 10 μM) for 24 h. Following the treatments, the survival of dopaminergic neurons were assessed by TH-immunostaining.

Data Analysis:

Data were analyzed with Prism 4.0 software (GraphPad Software, San Diego, Calif.). Dunnett post-hoc multiple comparison testing was used, if appropriate, to delineate significance between treated groups and the untreated samples. Differences with *p<0.05 and **p<0.01 were considered significant. Results are from at least two independent experiments performed in triplicate. The 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine and pyrazolo[3,4-b]pyridine exhibited IC₅₀ between 0.7 to 3 μM activity on these cell lines

Summary of Cell Culture Findings:

Several compounds in the claimed series dose-dependently protected against MPP+-induced cytotoxic cell death in N27 dopaminergic neuronal cells, dose-dependently protected against MPP+-induced increase in caspase-3 activity in N27 cells. Increase in caspase-3 activity correlates directly with apoptotic cell death. These series compounds also protected TH neurons from MPP+-induced dopaminergic neurodegeneration. Thus these compounds exhibit promising neuroprotective effect in cell culture models of PD; further validation of in an animal model of PD is undertaken.

In Vivo Models of Parkinsons Disease

Neuroprotective Effect of Orally Administered Series in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-Mouse Model of Parkinson's Disease (PD):

32 C57 black mice (C57/bl6, 6-8 week old, 25-30 g) were purchased from Charles River Laboratories, Ohio. Animals were divided into 4 groups of 8 animals each, group A for saline-treatment, group B for MPTP-treatment, group C for compounds 13, 13 HCl, 71, 105, 134 and 148, (10 mg/kg)+MPTP and group D for compounds 13, 13 HCl, 71, 105, 134 and 148 (30 mg/kg)+MPTP. All animals were housed under standard conditions: constant temperature (22±1° C.), humidity (relative, 30%), and a 12-h light/dark cycle. Mice were allowed free access to food and water. Use of the animals and protocol procedures were approved and supervised by the Committee on Animal Care at Iowa State University (Ames, Iowa). The test compounds 13, 13 HCl, 71, 105, 134 and 148 were dissolved in sterile water for this study. Prior to treatment animals were pre-trained on rotarod for at least 3 minutes every day for three successive days. For the pre-treatment study, test compounds 13, 13 HCl, 71, 105, 134 and 148 were administered once by oral gavage to group C animals (10 mg/kg) and group D animals at 30 mg/kg, 24 h prior to MPTP-injection and continued daily for additional 11 days. MPTP was dissolved in PBS and injected intraperitoneally (i.p.) at doses of 25 mg/kg, once daily for 5 days to group B, C and D animals. Group A animals were injected with saline (i.p.) to be used as controls. Urine and feces were collected on Day 8, behavioural studies were performed on Day 9 (4-days post-MPTP). On day 12 (7 days post-MPTP), 4 animals/group were sacrificed, different brain regions including striatum and substantia nigra were dissected. Left striatum were used for neurochemical analysis. Other 4 animals/group were perfused and used for TH immunohistological studies. Reference: Ghosh et al., Free Radic Biol Med. 2010 Dec. 1; 49(11):1674-84. Zhang et al., J Pharmacol Exp. Ther., 2007 September; 322(3):913-22, both of which are incorporated by reference herein in their entireties.

Orally Administered Test Compounds 13, 13 HCl, 71, 105, 134 and 148 Improve Locomotor Activities in MPTP-Injected Mice:

MPTP-treatment reduced the time-spent on the rotarod by 44% compared to saline-treated animals, whereas test compounds at 10 mg/kg increased the time spent by MPTP-treated mice to 101% suggesting that test compounds at 10 mg/kg completely restored MPTP-induced motor deficits to that of saline-treated animals. Similarly, test compounds at 30 mg/kg increased the time spent by MPTP-treated mice to 89% suggesting that test compounds at 30 mg/kg almost completely restored MPTP-induced motor deficits to that of saline-treated animals. Together, these results suggest that test compounds significantly restored MPTP induced motor co-ordination impairments.

Orally Administered Test Compounds 13, 13 HCl, 71, 105, 134 and 148 Restore Striatal Dopamine Levels in MPTP Treated Mice:

MPTP treatment decreased the striatal dopamine levels by 59% compared to that of saline-treated animals, whereas test compounds at 10 mg/kg and 30 mg/kg increased the dopamine levels in the presence of MPTP to 71% and 106% respectively suggesting that test compounds dose-dependently restored MPTP-induced dopamine loss. Together, these results suggest that test compounds rescues MPTP-induced neurochemical depletion in animal models of PD.

Orally Administered Test Compounds 13, 13 HCl, 71, 105, 134 and 148 Protect Against MPTP-Induced TH Neuronal Loss in the Substantia Nigra and in the Striatum:

Seven days after MPTP treatment, brains from 4 groups were harvested and grouped into sets 1-4 as described in the method section. Nigral and striatal sections were then subject to tyrosine hydroxylase (TH) immunostaining. MPTP treatment led to degeneration of TH positive dopaminergic neurons and its terminals in the striatum (2×), substantia nigra at lower (2×) and higher magnification (10×) compared to saline-treated animals, clearly visualized severe and significant loss of neurons in substantia nigra pars compacta (SNpc), substantia nigra lateralis and substantia nigra reticularis regions of nigral tract in MPTP-treated animals compared to saline-treated animals. Importantly, test compound 13, 13 HCl, 71, 105, 134 and 148 at 10 mg/kg and 30 mg/kg offered dose-dependent protection against MPTP-induced TH cell loss and dopaminergic degeneration both in the substantia nigra and in the striatum.

Intravenous Administration of Test Compounds 13, 13 HCl, 71, 105, 134 and 148:

24 C57 black mice (C57/bl6, 6-8 week old, and 25-30 g) were purchased from Charles River Laboratories, Ohio. Animals were divided into 3 groups of 8 animals each, group A for saline-treatment, group B for MPTP-treatment and group C compounds 13, 13 HCl, 71, 105, 134 and 148, (5 mg/kg)+MPTP. Compounds 13, 13 HCl, 71, 105, 134 and 148 were dissolved in PBS pH7.4 for this study. Prior to treatment animals were pre-trained on rotarod for at least 3 minutes every day for three successive days. Test compound (5 mg/kg) was administered intravenously to group C animals, 24 h prior to MPTP-injection and continued daily for additional 11 days. MPTP was dissolved in PBS and injected intraperitoneally (i.p.) at doses of 25 mg/kg, once daily for 5 days to group B and C animals. Group A animals were injected with saline (i.p.) to be used as controls. Urine and Faeces were collected on Day 8, behavioural studies were performed on Day 9 (4-days post-MPTP). On day 12 (7 days post-MPTP), 4 animals/group were sacrificed, different brains regions including striatum and substantia nigra were dissected. Left striatum were used for neurochemical analysis. Other 4 animals/group were perfused and used for TH immunohistological studies.

Intravenously Administered Test Compound Improves Locomotor Activities in MPTP-Injected Mice:

MPTP-treatment reduced the time spent on the rotarod by 53% compared to saline-treated animals, whereas test compounds at 10 mg/kg increased the time spent by MPTP-treated mice to 102% suggesting that test compounds at 5 mg/kg, i.v. completely restored MPTP-induced motor deficits to that of saline-treated animals. These results suggest that intravenous test compounds administration significantly restored MPTP induced motor co-ordination impairments.

Intravenously Administered Test Compounds Restore Striatal Dopamine Levels in MPTP Treated Mice:

MPTP-treatment decreased the striatal dopamine levels by 60% compared to that of saline-treated animals, whereas test compounds at 5 mg/kg restored dopamine levels in the presence of MPTP to 85%, nearly to that of saline treated animals. Together, these results suggest that test compounds rescues MPTP-induced neurochemical depletion in animal models of PD.

Intravenously Administered Compounds 13 HCl, 71, 105 and 134 Protect Against MPTP-Induced TH Neuronal Loss in the Substantia Nigra and in the Striatum:

MPTP treatment led to degeneration of TH positive dopaminergic neurons and its terminals in the striatum (2×), substantia nigra at lower (2×) and higher magnification (10×) compared to saline-treated animal clearly visualized severe and significant loss of neurons in substantia nigra pars compacta (SNpc), substantia nigra lateralis and substantia nigra reticularis regions of nigral tract in MPTP-treated animals. Importantly, test compounds at 5 mg/kg offered an almost a complete protection against MPTP-induced TH cell loss and dopaminergic degeneration both in the substantia nigra and in the striatum.

TABLE 4 BBB Pre-treatment results Group 4 Concentrations of test compounds in Mouse Sample ID Brain Homogenate (ng/gm) 1 0 ng/gm 2 5 ng/gm 3 10 ng/gm  4 11 ng/gm  5 2 ng/gm BQL = below quantitation limit of 1.00 ng/mL, whole brain/buffer homogenates Summary of Oral and Intravenous Pre-Treatment

The results indicate that both orally and intravenously administered 5-(pyrazin-2-yl)-1H-pyrazolo[3,4-b]pyridine, 5-(pyrazin-2-yl)-1H-pyrrolo[2,3-b]pyridine and pyrazolo[3, 4-b]pyridine derivatives improve behavioural functions, restore striatal dopamine levels and protect nigrostriatum in MPTP-treated mice. However, orally administered compounds were more effective than intravenously administered compounds in protecting against MPTP-induced motor deficits, dopamine depletion and TH neuronal loss. Together, these results strongly suggest that these compounds are effective neuroprotective agents in therapeutic intervention of PD. This data further supports the C57 black mice models are promising models for proof of concept studies targeting LRRK2 protein where its expression in mice shares ˜86% homology with the human protein NM-25730.

Post-Treatment Study

Neuroprotective Effect of Test Compounds 13, 13 HCl, 71, 105, 134 and 148 in MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine)-Mouse Model of Parkinson's Disease (PD):

32 C57 black mice (C57/bl6, 6-8 week old, 25-30 g) were purchased from Charles River Laboratories, Ohio. Animals were divided into 4 groups of 8 animals each, group A for saline-treatment, group B for MPTP-treatment, group C for MPTP+13 HCl, 71, 105 and 134 (30 mg/kg, oral) and group D for MPTP+13 HCl, 71, 105 and 134 (5 mg/kg, i.v.). All animals were housed in Laboratory of Animal Resources vivarium at Iowa State University under standard conditions: constant temperature (22±1° C.), humidity (relative, 30%), and a 12-h light/dark cycle. Mice were allowed free access to food and water. Use of the animals and protocol procedures were approved and supervised by the Committee on Animal Care at Iowa State University (Ames, Iowa). Test compounds were dissolved in PBS for this study. Prior to treatment animals were pre-trained on rotarod for at least 3 minutes every day for three successive days.

Group A animals were injected with saline (i.p.) to be used as controls. MPTP was dissolved in PBS and injected intraperitoneally (i.p.) at doses of 25 mg/kg, once daily for 5 days to group B, C and D animals. On day 4, prior to 4^(th) MPTP injection, group C animals received test compounds (30 mg/kg, oral) daily for eight days. Similarly, on day 4, prior to 4^(th) MPTP injection, group D animals received test compounds (5 mg/kg, i.v) daily for eight days. On day 9 (Post-test compounds administration) or day 12 (7 days post-MPTP), 4 animals/group were sacrificed, different brain regions including striatum and substantia nigra were dissected. Left striatum were used for neurochemical analysis. Other 4 animals/group were perfused and used for TH immunohistological studies.

TABLE 5 BBB Post-treatment results for 13 HCl PK20.155 Calc. Conc. (ng/g) of test Pooled Regions Subject ID compound 13 HCl in Mice Brains 70703 2.74 70704 3.03 70705 2.72 70706 2.92 70711 1.36 70712 2.32 70713 3.09 70714 3.79 Mean 2.75 % CV 25.5 70688 (control) BQL <1 ng/g Summary of Oral and Intravenous

Post-administration of test compounds 13 HCl, 105 and 148 by oral and i.v routes significantly protected against MPTP-induced motor-deficits and histological deficits and but not against neurochemical deficits.

Pharmacokinetics

Referring now to FIG. 2, mice PK: Mean Plasma Concentrations of test compounds 13 HCl, 71 and 105 in Mice Dosed 10 mg/kg PO and 5 mg/kg IV is shown. Table 6 reflects mice pharmacokinetics parameters.

TABLE 6 Mice Pharmacokinetics Parameters IV Dose (5 mg/kg) Vb Vss AUC0-¥ AUC0-last C₀ Cl t1/2 (L/ (L/ (h*ng/ (h*ng/ (ng/ (L/hr/ (hr) kg) kg) mL) mL) mL) kg) 10.6 87.1 45.2 882 787 193 5.67 Oral Dose (10 mg/kg) Cmax AUC0-¥ AUC0-last (ng/ Tmax t1/2 (h*ng/ (h*ng/ F mL) (hr) (hr) mL) mL) (%) 144 0.250 5.12 351 228 19.9

Rat PK:

To investigate the pharmacokinetic profiles of test compound following intravenous bolus administration and oral gavage in male Sprague Dawley rats.

TABLE 7 Rat Pharmacokinetics Parameters Route/Dose T_(max) C₀ C_(max) AUC_(0-t) AUC_(0-inf) T_(1/2) CL V_(ss) F (mg/kg) (h) (ng/mL) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (mL/min/kg) (L/kg) (%) IV (5) NA 902.15 ± 355.65 NA 1020.46 ± 646.82 685.61 2.26 122.84 15.15 — PO (30) 6.67 ± 2.31 NA 132.66 ± 40.84 1043.74 ± 497.14 NR NA NA NA 23.87

The plasma concentration-time data and plasma pharmacokinetic parameters of one of the compounds 13, 13 HCl, 71, 105, 134 and 148, following IV and PO administration in male Sprague Dawley rats are presented in Table 7. The individual and mean plasma concentration-time profiles following IV bolus administration of test compounds (5 mg/kg) to rats, the mean AUC_(0-t) and T_(1/2) were 1020.46 ng·h/mL and 2.26 h, respectively (Table 7) test compounds 13, 13 HCl, 71, 105, 134 and 148 showed clearance (CL) 122.84 mL/min/kg which is more than 100% of normal hepatic blood flow in rats. The mean apparent steady-state volume of distribution (V_(ss)) of compounds 13, 13 HCl, 71, 105, 134 and 148 was 15.15 L/kg, which is approximately 30-fold higher than the total body water normalized to body weight (i.e. 0.7 L/kg). Following oral administration of compounds 13, 13 HCl, 71, 105, 134 and 148 (30 mg/kg) solution, mean C_(max) was found to be 132.66 ng/mL and mean T_(max) was found to be 6.7 h. The mean AUC_(0-t) was found to be 1043.71 ng·h mL and the absolute oral bioavailability was found to be 24%.

Toxicology—In Vitro

Rat Hepatoma (H4IIE) Cell Line:

Cells were seeded into 96-well plates and cultured in medium containing 20% bovine serum. Following an equilibration period, cells were treated with compounds 13 and 13 HCl at concentrations of 0, 5, 10, and 50 μM for 24 hrs (overnight) at 37° C. in 5% CO₂. General cytotoxicity was evaluated by monitoring membrane integrity (MBR, cell supernatant), cell mass (PI) and mitochondrial function (cellular ATP levels). Rotenone was included as a positive control because it produces measurable effects in all three assays. DMSO at 0.5% in medium was included as a negative control. Three wells per exposure concentration were used in each assay. Results are summarized in Table 8 and are expressed as percent of control, with control values set at 100%. TC₅₀ values were calculated if the half-maximal response (50%) was achieved. Test compounds 13 and 13 HCl were rank-ordered from most to least toxic based on individual membrane integrity and ATP responses as well as resultant TC₅₀ values.

TABLE 8 Summary of the acute toxicity data Cell Mem Mem Tox % ATP Mass Tox Compound Solubility ATP % Control Cell Mass % Control Control TC₅₀ TC₅₀ TC₅₀ 5 μM 10 μM 50 μM 5 μM 10 μM 50 μM 5 μM 10 μM 50 μM 13 50 μM 99% 99% 74%  93% 88% 71% 100% 96% 94% >50 μM >50 μM >50 μM 13 HCl 50 μM 93% 91% 78%  89% 81% 62%  93% 91% 90% >50 μM >50 μM >50 μM 0.001 μM 0.1 μM 1 μM 0.001 μM 0.1 μM 1 μM 0.001 μM 0.1 μM 1 μM Rotenone  1 μM 98% 34%  4% 105% 54% 25% 100% 98% 46% 0.08 μm 0.23 μM 0.93 μM

Results Summary:

Test compounds 13 and 13 HCl were rank-ordered from highest to lowest probability of producing toxicity based first on membrane toxicity, then on ATP, and finally on cell mass (PI). It is important to note that within a given grouping compounds may be classified as to their primary effect being either cytotoxic, cytostatic, or reducing mitochondrial function. In general, compound responses above 80% of vehicle control are considered to be low-to-no effect, moderates generally rank 80% to 50% of vehicle control, and those compounds that cross the 50% mark are considered to have higher probability of toxicity.

Test Compounds 13 HCl and 148 Inhibition of Monoamine Oxidases (MAO-A and MAO-B)

Experimental Procedure:

Test compounds (0.01-100 μM) final DMSO concentration 0.1%, were incubated with recombinant human MAO-A (2 μg/mL) or MAO-B (2 μg/mL) in the presence of the probe substrate kynuramine (50 μM) for 25 min at 37° C. The non-selective MAO modulator, tranylcypromine, was screened alongside the test agents with tranylcypromine as a positive control. The reactions were terminated by addition of methanol containing internal standard for analytical quantification. The quenched samples were incubated at 4° C. for 10 min and centrifuged at 4° C. for 10 min. The supernatant was removed and analyzed by LC-MS/MS for the probe metabolite 4-hydroxyquinoline. A decrease in the formation of the metabolite compared to vehicle control was used to calculate an IC₅₀ value (the test concentration which produces 50% inhibition.

TABLE 9 MAO inhibition summary of IC₅₀ values MAO-A MAO-B Control Inhibitors IC₅₀ (μM) IC₅₀ (μM) Tranylcypromine 0.12 0.11 Compound 13 >100 >100 Compound 148 >100 8.8 Primary Biochemical Assay

Test compounds in Table 1 have been profiled in a standard in vitro compound selection program to detect potential adverse activity, additional unexpected activity, and relative selectivity and specificity to choose the “lead drug candidate” for further development. The screen covered 65 primary molecular targets, including adenosine, adrenergic, bradykinin, dopamine, glutamate, histamine, muscarinic, serotonin, opiate, and potassium channels including hERG. The test compounds in Table 1 at 10 μM did not inhibit the molecular targets greater than 50%.

Structural Homology Modeling—LRRK2 and G2019S

The compounds of Formula I, II and VII and in Table 1 and 2 were designed using a structural homology model of LRRK2 and its mutant form of LRRK2 G2019S. The homology model of structural model of the kinase domain of LRRK2 was constructed using Leucine-Rich Repeat Kinase (LRRK2) domain region sequence (1879-2140) from the full length protein sequence NP_(—)940980 (FIG. 3). Sequence search with in the RCSB provided the 5 homologues X-ray crystal structure templates with 30-32% sequence identity and ˜50% sequence similarities was considered as starting point for multiple sequence alignment using Clustal W alignment (FIG. 4) and homology modeling. The Swiss-Model was applied in sequence alignment, model building, loop prediction and refinement. With the application of FFDD (Fragment-Field Drug Design) workflow design strategy, the final models of LRRK2 shown in FIG. 5 were utilized and served as template for designing claimed compounds.

Based on the 3-D profile scoring the structural template chosen from PDB database and were both MLK and BRAF crystal structures. Several models were built and refined to check the 3D profile and are shown in FIG. 5.

General Administration and Pharmaceutical Compositions

When used as pharmaceuticals, the compounds disclosed herein are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared using procedures well known in the pharmaceutical art and include at least one compound disclosed herein. The compounds may also be administered alone or in combination with adjuvants that enhance stability of the compounds, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increased inhibitory activity, provide adjunct therapy, and the like. The compounds may be used on their own or in conjunction with other active substances, optionally also in conjunction with other pharmacologically active substances. In general, the compounds may be administered in a therapeutically or pharmaceutically effective amount, but may be administered in lower amounts for diagnostic or other purposes.

When the compounds of the present invention are administered to a human, the compound itself or the compound after admixing with a suitable pharmacologically acceptable carrier, excipient, diluent and the like can be administered orally or parenterally as a safe pharmaceutical composition, such as an oral administration agent (e.g., powder, granule, tablet, capsule etc.), a parenteral administration agent (e.g., injection, an external preparation (e.g., nasal preparation, percutaneous preparation etc.)), a suppository (e.g., rectal suppository, vaginal suppository and the like) and the like.

Thus, administration can be, for example, orally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. The pharmaceutical compositions will generally include a conventional pharmaceutical carrier and/or excipient and a compound as disclosed herein as the/an active agent, and, in addition, may include other agents such as but not limited to medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, vehicles, or combinations thereof. Such pharmaceutically acceptable excipients, carriers, or additives as well as methods of making pharmaceutical compositions for various modes or administration are well-known to those of skill in the art. The state of the art is evidenced, e.g., by Remington: The Science and Practice of Pharmacy, 20th Edition, A. Gennaro (ed.), Lippincott Williams & Wilkins, 2000; Handbook of Pharmaceutical Additives, Michael & Irene Ash (eds.), Gower, 1995; Handbook of Pharmaceutical Excipients, A. H. Kibbe (ed.), American Pharmaceutical Ass'n, 2000; and H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger, 1990; each of which is incorporated herein by reference in their entireties to better describe the state of the art. It will be noted that in all of the pharmaceutical compositions enumerated herein, the compounds of the invention are formulated with an acceptable carrier or excipient. The carriers or excipients used must, of course, be acceptable in the sense of being compatible with the other ingredients of the composition and must not be deleterious to the recipient. The carrier or excipient can be a solid or a liquid, or both, and is preferably formulated with the compound of the invention as a unit-dose composition, for example, a tablet. Such carriers or excipients include inert fillers or diluents, binders, lubricants, disintegrating agents, solution retardants, resorption accelerators, absorption agents, and coloring agents. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, polyethylene glycol, waxes, and the like. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Examples of a coating agent include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, pluronic F68, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (Rohm Pharm GmbH, Germany, a copolymer of methacrylic acid with acrylic acid), pigment (e.g., titanium oxide, bengara etc.) and the like.

As one of skill in the art would expect, the forms of the compounds of the invention utilized in a particular pharmaceutical formulation will be selected that possess suitable physical characteristics (e.g., water solubility) that is required for the formulation to be efficacious.

The content of the selected active compound of the present invention in the preparation varies depending on the dosage form. For example, the content thereof in the aforementioned oral administration agent is about 1 to about 99 wt %, preferably about 10 to 99 wt %, more preferably about 10 to about 95 wt %. In the aforementioned parenteral administration agent, for example, it is about 0.001 to about 99 wt %, preferably about 0.001 to about 95 wt %. The content of the components other than the active compound in the preparation is generally about 1 to 99.999 wt %, preferably about 10 to about 90 wt %. For injection, for example, an aqueous injection can be prepared using a compound of the present invention together with a solubilizer (e.g., beta.-cyclodextrins etc.), a dispersing agent (e.g., Tween 80 (Atlas Powder Co., USA), HCO60 (Nikko Chemicals Co., Ltd.), carboxymethylcellulose, sodium alginate etc.), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol etc.), an isotonic agent (e.g., sodium chloride, glycerine, sorbitol, glucose etc.) and the like according to a conventional method. Alternatively, an oily injection can be formed by dissolving, suspending or emulsifying as appropriate the compound in a vegetable oil (e.g., olive oil, sesame oil, peanut oil, cottonseed oil, corn oil etc.), propylene glycol and solid like dosage forms for oral administration of the compounds include capsules, tablets, pills, powders, and granules. For such oral administration, a pharmaceutically acceptable composition containing a compound(s) of the invention is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, pregelatinized starch, magnesium stearate, sodium saccharine, talcum, cellulose ether derivatives, glucose, gelatin, sucrose, citrate, propyl gallate, and the like.

Such solid pharmaceutical formulations may include formulations, as are well-known in the art, to provide prolonged or sustained delivery of the drug to the gastrointestinal tract by any number of mechanisms, which include, but are not limited to, pH sensitive release from the dosage form based on the changing pH of the small intestine, slow erosion of a tablet or capsule, retention in the stomach based on the physical properties of the formulation, bioadhesion of the dosage form to the mucosal lining of the intestinal tract, or enzymatic release of the active drug from the dosage form.

Liquid dosage forms for oral administration of the compounds include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs, optionally containing pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like. These compositions can also contain additional adjuvants such as wetting, emulsifying, suspending, sweetening, flavoring, and perfuming agents.

A solid external agent can be produced from the compound of the present invention as it is, or by adding an excipient (e.g., glycol, mannitol, starch, crystalline cellulose etc.), a thickener (e.g., natural gums, cellulose derivative, acrylic polymer etc.) and the like, mixing the same and preparing into a powdery composition. A semi-solid external agent can be produced by a conventional method and preferably used as an aqueous or oily gel, or an ointment. A liquid external preparation can be produced by preparing into an oily or aqueous suspension according to a method employed for production of injection or a similar method. In addition, a pH-adjusting agent (e.g., carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide etc.), a preservative (e.g., p-oxybenzoic acid esters, chlorobutanol, benzalkonium chloride etc.) and the like may be added as appropriate to a solid, semi-solid or liquid external agent. To be specific, for example, an ointment can be prepared, which contains a compound of the present invention in an amount of about 0.1 to about 100 mg per 1 g, using petrolatum, lanolin and the like as a base material. Topical dosage forms of the compounds include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, eye ointments, eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. Topical application may be once or more than once per day depending upon the usual medical considerations. The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation, preferably they will form up to about 80% of the formulation.

Formulations including a compound of the present invention can be also prepared as an oily or aqueous solid, semi-solid or liquid suppository. The oily base used as appropriate for producing suppository may be, for example, hard fat, higher fatty acid glyceride (e.g., cacao butter, Witepsols (Dynamitnobel Ltd.) etc.), middle fatty acid (e.g., migriol acid (Dynamitnobel Co., Ltd.) etc.), vegetable oil (e.g., sesame oil, soybean oil, cottonseed oil etc.) and the like. As an aqueous base, for example, polyethylene glycols, propylene glycol and the like may be used, and as an aqueous gel base, for example, natural gums, cellulose derivative, vinyl polymer, acrylic polymer and the like may be used as appropriate.

While the dose of a compound of the present invention varies depending on the age, body weight, condition, dosage form, administration method, administration period and the like, for example, it is generally about 0.01 to about 1000 mg/kg, preferably about 0.01 to about 100 mg/kg, more preferably about 0.1 to about 100 mg/kg, particularly about 0.1 to about 50 mg/kg, specifically about 1.5 to about 30 mg/kg per day, as an amount of a compound of the present invention, for one patient (adult, body weight about 60 kg) with AD, PD or disease or condition as enumerated herein, which is orally or parenterally administered once to several times a day. The dose varies depending on various conditions as mentioned above, and in some cases an amount smaller than the aforementioned dose is sufficient and in other cases administration beyond the above range may be necessary.

Examples of Dosage Forms

Treatment of Parkinson's disease symptoms Dosage Recommended: 2 capsules 2 times daily Active Compound 13 90 mg

Treatment of Alzheimer's disease symptoms Dosage Recommended: 2 capsules 3 times daily Active Compound 13 90 mg Examples of Formulations

A. TABLETS Amount per tablet Component (mg) active substance 400 lactose 40 corn starch 40 polyvinylpyrrolidone 15 magnesium stearate 5 TOTAL 500

The finely ground active substance, lactose, and some of the corn starch are mixed together. The mixture is screened, and then moistened with a solution of polyvinylpyrrolidone in water, kneaded, wet-granulated and dried. The granules, the remaining corn starch and the magnesium stearate are screened and mixed together. The mixture is compressed to produce tablets of suitable shape and size.

B. TABLETS Amount per tablet Component (mg) active substance 75 lactose 5 corn starch 5 polyvinylpyrrolidone 4 magnesium stearate 1 microcrystalline cellulose 5 sodium-carboxymethyl 5 starch TOTAL 100

The finely ground active substance, some of the corn starch, lactose, microcrystalline cellulose, and polyvinylpyrrolidone are mixed together, the mixture is screened and worked with the remaining corn starch and water to form a granulate which is dried and screened. The sodium-carboxymethyl starch and the magnesium stearate are added and mixed in and the mixture is compressed to form tablets of a suitable size.

C. COATED TABLETS Amount per tablet Component (mg) active substance 65 Lactose 10 corn starch 11.5 polyvinylpyrrolidone 3 magnesium stearate 0.5 TOTAL 90

The active substance, corn starch, lactose, and polyvinylpyrrolidone are thoroughly mixed and moistened with water. The moist mass is pushed through a screen with a 1 mm mesh size, dried at about 45° C. and the granules are then passed through the same screen. After the magnesium stearate has been mixed in, convex tablet cores with a diameter of 6 mm are compressed in a tablet-making machine. The tablet cores thus produced are coated in known manner with a covering consisting essentially of sugar and talc. The finished coated tablets are polished with wax.

D. CAPSULES Amount per capsule Component (mg) active substance 90 corn starch 8.5 magnesium stearate 1.5 TOTAL 100

The substance and corn starch are mixed and moistened with water. The moist mass is screened and dried. The dry granules are screened and mixed with magnesium stearate. The finished mixture is packed into size 1 hard gelatin capsules.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

REFERENCES

The following references, as well as all references cited in this specification, are incorporated by reference herein in their entirety:

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What is claimed is:
 1. A compound having the structure of Formula I:

wherein: X is independently —N; L₁ is independently a substituted or unsubstituted 6-membered aryl, heteroaryl, substituted or unsubstituted 5-membered aryl or heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, —C(O)_(n), S(O)n, —O, —NH, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted 2 to 5 membered heteroalkylene groups; where “n” is from 0 to 2, —CH₃, halo, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, —OR₄ —SR₄, —NHR₄, —C(O)R₄—, —C(S)R₄, —C(O)NR₄, and —S(O)₂R₄, —S(O)₂NHR₄, —NHCOR₄, —CONHR₄, CONR₄, —CH═CHR₄, —NCH₃R₄, R₄ is optionally substituted at each occurrence and is selected from alkyl, heteroaryl, cycloalkyl, heterocycloalkyl, —C₁₋₆ alkyl, —C₁₋₄ haloaliphatic, —C₂₋₆ alkenyl, mono/dialkylamino, aryl C₄₋₇ heterocycloakyl, —C₅₋₆ aryl and, wherein the functional groups are each optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted lower alkyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino functional groups or additional substituents selected from —NR₅R₆ and —CR₅R₆ respectively, and wherein R₄ together with the carbon to which it is attached may form a 3-7 member monocyclic cycloalkyl, 5-6 membered monocyclic heterocycloalkyl, heterocycloalkyl, heteroaryl; wherein the monocyclic cycloalkyl or heterocycloalkyl are each independently optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted lower alkyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino; R₅ and R₆ are each independently —H, cycloalkyl, alkyl, heterocycloalkyl, aryl, heteroaryl, —C₁₋₆ alkyl, —C₃₋₇ cycloalkyl, —C₃₋₆ heterocyclocycloalkyl, each optionally substituted with one or more of fluoro, —NH₂, hydroxyl, fluoro substituted C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, alkoxy, alkylthio, mono/dialkylamino and cycloalkylamino; wherein L1 is optionally substituted with —R₇ or Y₉; wherein R₇ is independently a bond, substituted or unsubstituted —C═S, C═O, —C₁₋₆ alkyl, each optionally further substituted by C₁₋₆ alkyl, (for example, (—CH₂)_(n), wherein n is an integer from 0-3), —O, —S, —SO, SO₂, —NH or —CH═CH groups; and R₇ may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, wherein C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl are each optionally substituted with one or more of fluoro, —OH, —NH₂, OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, mono/dialkylamino, and alkyl carbon bound to the N of —C(O)NHR₈, —C(S)NHR₈ or —S(O)₂NHR₈; wherein the alkyl chain(s) of OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, and mono/dialkylamino are each optionally independently substituted with one or more of fluoro, —OH, —NH₂, wherein any substitution of the alkyl chain carbon bound to O of alkoxy, S of thioalkyl or N of mono/dialkylamino is fluoro; R₈ combines with the nitrogen to which it is attached to form a 5-7 member heterocycloalkyl optionally substituted with one or more of fluoro, —OH, —NH2, C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, fluoro substituted C₁₋₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, OCH₃, CH₂OCH₃, fluoro substituted OCH₃, CH₂OCH₃, methylthio, ethylthio, butylthio, isobutylthio, and fluoro-substituted methylthio, ethylthio, butylthio, isobutylthio; Y₉ is —C₁₋₆ alkyl, C₃₋₇ cycloalkyl, —C₁₋₆-alkyl —C₃₋₇-cycloalkyl, —C₁₋₆ alkyl heteroaryl, —C₄₋₇ heterocycloalkyl, aryl or heteroaryl, each optionally independently substituted with one or more substituents selected from R₄; and R₁, R₂ and R₃ are each independently halo, —CN, —NH₂, —C₁₋₆ aliphatic, —OC₁₋₆ aliphatic, —C₃₋₆ cycloaliphatic, —OC₃₋₆ cycloaliphatic, —NH₂, —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, —CONH₁₋₆ heterocycloalkyl, —NHC₁₋₆ aliphatic, —NHC₃₋₆ cycloaliphatic, —NHS(O)₂C₁₋₆ aliphatic, —NHS(O)₂C₃₋₆ cycloaliphatic, —NHS(O)₂ phenyl, —NHS(O)₂ benzyl, —NHS(O)₂ heteroaryl, —S(O)₂C₁₋₆ aliphatic, —S(O)₂C₃₋₆ cycloaliphatic, —S(O)₂ phenyl, —S(O)₂ benzyl, —S(O)₂ heteroaryl, —S(O)₂NHC₁₋₆ aliphatic, —S(O)₂NHC₃₋₆ cycloaliphatic, —S(O)₂NHphenyl, —S(O)₂NHbenzyl, or —S(O)₂NHheteroaryl, wherein each of said heteroaryl of R₁, R₂ and R₃ is independently a 5- or 6-member ring having 1, 2, or 3 atoms selected from N, O, or S, and wherein said aliphatic, cycloaliphatic, phenyl, benzyl, or heteroaryl of R₁, R₂ and R₃ is each optionally substituted with 1, 2, or 3 ZR₁₀ groups; wherein one or two of R₁, R₂ and R₃ may be hydrogen when one or two of R₁, R₂ and R₃ are halo, —CN, —NH₂, —C₁₋₆ aliphatic, —OC₁₋₆ aliphatic, —C₃₋₆ cycloaliphatic, —OC₃₋₆ cycloaliphatic, —NH₂, —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, —CONH₁₋₆ heterocycloalkyl, —NHC₁₋₆ aliphatic, —NHC₃₋₆ cycloaliphatic, —NHS(O)₂C₁₋₆ aliphatic, —NHS(O)₂C₃₋₆ cycloaliphatic, —NHS(O)₂ phenyl, —NHS(O)₂ benzyl, —NHS(O)₂ heteroaryl, —S(O)₂C₁₋₆ aliphatic, —S(O)₂C₃₋₆ cycloaliphatic, —S(O)₂ phenyl, —S(O)₂ benzyl, —S(O)₂ heteroaryl, —S(O)₂NHC₁₋₆ aliphatic, —S(O)₂NHC₃₋₆ cycloaliphatic, —S(O)₂NHphenyl, —S(O)₂NHbenzyl, or —S(O)₂NHheteroaryl, wherein each of said heteroaryl of R₁, R₂ and R₃ is independently a 5- or 6-member ring having 1, 2, or 3 atoms selected from N, O, or S, and wherein said aliphatic, cycloaliphatic, phenyl, benzyl, or heteroaryl of R₁, R₂ and R₃ is each optionally substituted with 1, 2, or 3 ZR₁₀ groups; wherein ZR₁₀ is halo, oxo, —C₁₋₂ alkyl, substituted C₁₋₂ alkyl with 1-3 —F or —Cl atoms, —C₃₋₆ cycloalkyl, —OH, —OC₁₋₂ alkyl, —OC₁₋₂ alkyl substituted with 1-3 —F or —Cl atoms, —C(O)C₁₋₂ alkyl, or —SC₁₋₂ alkyl, and the pharmaceutically acceptable salts, isomers and tautomers thereof.
 2. The invention of claim 1 wherein L₁ is selected from the group consisting of

wherein

 indicates the attachment to L₁ of Formula I; wherein R is —H, halo, —CH₃, —CH₂CH₃, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, OH, —CH₂OH, —SO₂CH₃, SO₂N (CH₃)₂, —COCH₃; and wherein “n” is an integer from 0 to
 2. 3. The invention of claim 1 wherein R₁, R₂ and R₃ are each independently:

wherein

 indicates the attachment to R₁, R₂ and R₃ of Formula I.
 4. The invention of claim 1 wherein R₁, R₂ and R₃ at each occurrence is independently —H, optionally substituted C₁₋₆ alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl.
 5. The invention of claim 1 wherein R₁, R₂ and R₃ are each independently —NHCOC₁₋₆ alkyl, —NHCO₁₋₆ cycloalkyl, —NHCO₁₋₆ heterocycloalkyl, —CONHC₁₋₆ alkyl, —CONH₁₋₆ cycloalkyl, or —CONH₁₋₆ heterocycloalkyl.
 6. The invention of claim 1 wherein R₁, R₂ and R₃ are each independently directly substituted by —H, —F, —Cl, —OCF₃, CF₃, —CH₃, —OCHF₂ or —OCH₃, wherein at least one of R₁ R₂ and R₃ is not hydrogen.
 7. The invention of claim 1 wherein R₁, R₂ and R₃ are each independently formula III or IV

wherein

 indicates the attachment to Formula I of one of R₁, R₂ and R₃ positions and R₉ is —H, —CH₃, —CH(CH₃)₂, —CH₂CH₃, —CH₂CH₂—OH, or —C₃₋₆-alkyl.
 8. The invention of claim 1 comprising a compound of formula VIII wherein

indicates attachment to R₁, R₂ and R₃ of Formula I respectively

wherein R₁₂ is —H, halo, —CH₃, —CH₂CH₃, —OCH₃, —CF₃, OCF₃, —CN, —NH₂, —CH₂OCF₂, —COOH, OH, —CH₂OH, —SO₂CH₃, SO₂N (CH₃)₂, or —COCH₃.
 9. A compound according to claim 1 which is a protein kinase inhibitor.
 10. A compound according to claim 9 wherein the compound is an inhibitor of a protein kinase selected from the group consisting of ABL1 T315I (SEQ ID NO 1), ALK2 (SEQ ID NO 2), ARK5 (SEQ ID NO 64), Aurora A (SEQ ID NO 3), Aurora B (SEQ ID NO 4), Aurora C (SEQ ID NO 5), BRAF (SEQ ID NO 6), BRAF V599E (SEQ ID NO 7), CHK1 (SEQ ID NO 8), CHK2 (SEQ ID NO 9), C-KIT (SEQ ID NO 10), CLK1 (SEQ ID NO 11), CLK2 (SEQ ID NO 12), CLK3 (SEQ ID NO 13), CLK4 (SEQ ID NO 14), CSF1R (SEQ ID NO 15), DYRK1A (SEQ ID NO 16), DYRK1B (SEQ ID NO 17), DYRK2 (SEQ ID NO 18), DYRK3 (SEQ ID NO 19), DYRK4 (SEQ ID NO 20), FLT3 (SEQ ID NO 21), GSK3β (SEQ ID NO 22), JAK1 (SEQ ID NO 23), JAK2 (SEQ ID NO 24), JAK3 (SEQ ID NO 25), KDR (SEQ ID NO 26), LCK (SEQ ID NO 27), LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), LRRK2 T1602S R1628P T2352M (SEQ ID NO 29), MAP4K4 (SEQ ID NO 30), MELK (SEQ ID NO 31), MLK (SEQ ID NO 32), PDGFR (SEQ ID NO 41), PKA (SEQ ID NO 63), PKCα (SEQ ID NO 42), PKCτ (SEQ ID NO 43), PIM1 (SEQ ID NO 44), RET (SEQ ID NO 45), ROCK1 (SEQ ID NO 46), ROCK2 (SEQ ID NO 47), RSK1 (SEQ ID NO 48), RSK2 (SEQ ID NO 49), p70S6K (SEQ ID NO 50), SGK1 (SEQ ID NO 51), SNF1LK (SEQ ID NO 52), SIK2 (SEQ ID NO 53), SYK (SEQ ID NO 54), TNIK (SEQ ID NO 55), TRKA (SEQ ID NO 56), TRKB (SEQ ID NO 57) and TRKC (SEQ ID NO 58) and mutations thereof.
 11. A compound according to claim 9 wherein the compound is an inhibitor of a protein kinase selected from the group consisting of LRRK2 (SEQ ID NO 62), LRRK2 G2019S (SEQ ID NO 28), CHK1 (SEQ ID NO 8) and DYRK1A (SEQ ID NO 16) and mutations thereof.
 12. A compound according to claim 9 wherein the compound is an inhibitor of at least one of LRRK2 T1602S R1628P and T2352M (SEQ ID NO 29) variants.
 13. A composition comprising a compound according to claim 1 and at least one pharmaceutically acceptable carrier.
 14. A method of making a compound of Formula I according to claim 1 comprising: 